Compounds and pharmaceutical compositions thereof for the treatment of inflammatory disorders

ABSTRACT

Novel benzimidazoles according to Formula I, able to inhibit JAK are disclosed, these compounds may be prepared as a pharmaceutical composition, and may be used for the prevention and treatment of a variety of conditions in mammals including humans, including by way of non-limiting example, allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferons.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a Divisional of co-pending U.S.Non-Provisional application Ser. No. 14/599,644 filed Jan. 19, 2015,which in turn, claims the priority of co-pending United KingdomApplication No. GB1401086.2 filed on Jan. 23, 2014. Applicants claim thebenefits of said United Kingdom Application under 35 U.S.C. § 119 andthe said U.S. Non-Provisional application under 35 U.S.C. § 120, and thedisclosures of all of said applications are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compounds that are inhibitors of JAK, afamily of tyrosine kinases that are involved in allergic diseases,inflammatory diseases, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or hypersecretion of interferons. Inparticular, the compounds of the invention inhibit JAK1 and/or TYK2. Thepresent invention also provides methods for the production of thecompounds of the invention, pharmaceutical compositions comprising thecompounds of the invention, methods for the prevention and/or treatmentof diseases involving allergic diseases, inflammatory diseases,autoimmune diseases, proliferative diseases, transplantation rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or hypersecretion of interferons by administering a compound ofthe invention.

BACKGROUND OF THE INVENTION

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transducecytokine signaling from membrane receptors to STAT transcriptionfactors. Four JAK family members are described, JAK1, JAK2, JAK3 andTYK2. Upon binding of the cytokine to its receptor, JAK family membersauto- and/or transphosphorylate each other, followed by phosphorylationof STATs that then migrate to the nucleus to modulate transcription.JAK-STAT intracellular signal transduction serves the interferons, mostinterleukins, as well as a variety of cytokines and endocrine factorssuch as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL. (Vainchenker etal., 2008)

The combination of genetic models and small molecule JAK inhibitorresearch revealed the therapeutic potential of several JAKs. JAK3 isvalidated by mouse and human genetics as an immune-suppression target.(O'Shea et al., 2004) JAK3 inhibitors were successfully taken intoclinical development, initially for organ transplant rejection but lateralso in other immuno-inflammatory indications such as rheumathoidarthritis (RA), psoriasis and Crohn's disease(http://clinicaltrials.gov/).

TYK2 is a potential target for immuno-inflammatory diseases, beingvalidated by human genetics and mouse knock-out studies. (Levy andLoomis, 2007)

JAK1 is a target in the immuno-inflammatory disease area. JAK1heterodimerizes with the other JAKs to transduce cytokine-drivenpro-inflammatory signaling. Therefore, inhibition of JAK1 is of interestfor immuno-inflammatory diseases with pathology-associated cytokinesthat use JAK1 signaling, such as IL-6, IL-4, IL-5, IL-13, or IFNgamma,as well as for other diseases driven by JAK-mediated signaltransduction.

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of an RA therapy, therefore, is not only to slow down the diseasebut to attain remission in order to stop the joint destruction. Besidesthe severity of the disease outcome, the high prevalence of RA (˜0.8% ofadults are affected worldwide) means a high socio-economic impact. (Choyand Panayi, 2001; Firestein, 2003; Lee and Weinblatt, 2001; O'Dell,2004; Smolen and Steiner, 2003).

JAK1 and JAK2 are implicated in intracellular signal transduction formany cytokines and hormones. Pathologies associated with any of thesecytokines and hormones can be ameliorated by JAK1 and JAK2 inhibitors.Hence, several allergic or inflammatory conditions and autoimmunediseases might benefit from treatment with compounds described in thisinvention including rheumatoid arthritis, systemic lupus erythematosis,juvenile idiopathic arthritis, osteoarthritis, asthma, chronicobstructive pulmonary disease COPD, tissue fibrosis, eosinophilicinflammation, esophagitis, inflammatory bowel diseases (e.g. Crohn's,ulcerative colitis), transplantation, graft-versus-host disease,psoriasis, myositis, multiple sclerosis. (Kopf et al., 2010)

Osteoarthritis (also referred to as OA, or wear-and-tear arthritis) isthe most common form of arthritis and is characterized by loss ofarticular cartilage, often associated with hypertrophy of the bone andpain. (Wieland et al., 2005)

Osteoarthritis is difficult to treat. At present, no cure is availableand treatment focuses on relieving pain and preventing the affectedjoint from becoming deformed. Common treatments include the use ofnon-steroidal anti-inflammatory drugs (NSAIDs). Although dietarysupplements such as chondroitin and glucosamine sulphate have beenadvocated as safe and effective options for the treatment ofosteoarthritis, a recent clinical trial revealed that both treatmentsdid not reduce pain associated with osteoarthritis. (Clegg et al., 2006)

Stimulation of the anabolic processes, blocking catabolic processes, ora combination of these two, may result in stabilization of thecartilage, and perhaps even reversal of the damage, and thereforeprevent further progression of the disease. Therapeutic methods for thecorrection of the articular cartilage lesions that appear during theosteoarthritic disease have been developed, but so far none of them havebeen able to mediate the regeneration of articular cartilage in situ andin vivo. Taken together, no disease modifying osteoarthritic drugs areavailable.

Vandeghinste et al. (Vandeghinste et al., 2005) discovered JAK1 as atarget whose inhibition might have therapeutic relevance for severaldiseases including OA. Knockout of the JAK1 gene in mice demonstratedthat JAK1 plays essential and non-redundant roles during development:JAK1−/− mice died within 24 h after birth and lymphocyte development wasseverely impaired. Moreover, JAK1 −/− cells were not, or less, reactiveto cytokines that use class II cytokine receptors, cytokine receptorsthat use the gamma-c subunit for signaling and the family of cytokinereceptors that use the gp130 subunit for signaling. (Rodig et al., 1998)

Various groups have implicated JAK-STAT signaling in chondrocytebiology. Li et al. (Li et al., 2001) showed that Oncostatin M inducesMMP and TIMP3 gene expression in primary chondrocytes by activation ofJAK/STAT and MAPK signaling pathways. Osaki et al. (Osaki et al., 2003)showed that interferon-gamma mediated inhibition of collagen II inchondrocytes involves JAK-STAT signaling. IL1-beta induces cartilagecatabolism by reducing the expression of matrix components, and byinducing the expression of collagenases and inducible nitric oxidesynthase (NOS2), which mediates the production of nitric oxide (NO).Otero et al. (Otero et al., 2005) showed that leptin and IL 1-betasynergistically induced NO production or expression of NOS2 mRNA inchondrocytes, and that that was blocked by a JAK inhibitor. Legendre etal. (Legendre et al., 2003) showed that IL6/IL6 Receptor induceddownregulation of cartilage-specific matrix genes collagen II, aggrecancore and link protein in bovine articular chondrocytes, and that thiswas mediated by JAK/STAT signaling. Therefore, these observationssuggest a role for JAK kinase activity in cartilage homeostasis andtherapeutic opportunities for JAK kinase inhibitors.

JAK family members have been implicated in additional conditionsincluding myeloproliferative disorders (O'Sullivan et al., 2007), wheremutations in JAK2 have been identified. This indicates that inhibitorsof JAK in particular JAK2 may also be of use in the treatment ofmyeloproliferative disorders. Additionally, the JAK family, inparticular JAK1, JAK2 and JAK3, has been linked to cancers, inparticular leukaemias e.g. acute myeloid leukaemia (O'Sullivan et al.,2007; Xiang et al., 2008), and acute lymphoblastic leukaemia (Mullighanet al., 2009) or solid tumours e.g. uterine leiomyosarcoma(Constantineseu et al., 2008), prostate cancer (Tam et al., 2007). Theseresults indicate that inhibitors of JAK, in particular of JAK1 and/orJAK2, may also have utility in the treatment of cancers (leukaemias andsolid tumours e.g. uterine leiomyosarcoma, prostate cancer).

In addition, Castleman's disease, multiple myeloma, mesangialproliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma arelikely due to hypersecretion of the cytokine IL-6, whose biologicaleffects are mediated by intracellular JAK-STAT signalling (Naka et al.,2002). This result shows that inhibitors of JAK may also find utility inthe treatment of said diseases.

The current therapies are not satisfactory and therefore there remains aneed to identify further compounds that may be of use in the treatmentof allergic diseases, inflammatory diseases, autoimmune diseases,proliferative diseases, transplantation rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or hypersecretionof interferons. The present invention therefore provides compounds,methods for their manufacture and pharmaceutical compositions comprisingthe compounds of the invention together with a suitable pharmaceuticalcarrier. The present invention also provides for the use of a compoundof the invention in the preparation of a medicament for the treatment ofallergic diseases, inflammatory diseases, autoimmune diseases,proliferative diseases, transplantation rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or hypersecretionof interferons.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the compounds ofthe invention are able to act as inhibitors of JAK and that they areuseful for the treatment of allergic diseases, inflammatory diseases,autoimmune diseases, proliferative diseases, transplantation rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or hypersecretion of interferons. In a specific aspect thecompounds of the invention are inhibitors of JAK1 and/or TYK2. Thepresent invention also provides methods for the production of thesecompounds, pharmaceutical compositions comprising these compounds andmethods for treating allergic diseases, inflammatory diseases,autoimmune diseases, proliferative diseases, transplantation rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or hypersecretion of interferons by administering a compound ofthe invention.

Accordingly, in a first aspect of the invention, the compounds of theinvention are provided according to Formula (I):

wherein

-   R¹ is H, or Me;-   L₁ is —NR²—; —O—, or —CH₂—;-   Cy is phenyl, or 5-9 membered monocyclic or fused bicyclic    heteroaryl comprising 1 to 4 heteroatoms independently selected from    N, O, and S;-   R² is H, or C₁₋₄ alkyl;-   R³ is H, halo, C₁₋₄ alkyl optionally substituted with one or more    halo, or C₁₋₄ alkoxy optionally substituted with one or more halo;-   R⁴ is H, or halo;-   R⁵ is —CN, halo, or is -L₂-R⁶-   -L₂ is absent, or is —C(═O)—, —C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂—,    —SO₂NR⁷—, or —NR⁷SO₂—;-   R⁶ is H, or C₁₋₆ alkyl optionally substituted with one or more    independently selected R⁸ groups;-   R⁷ is H, or C₁₋₄ alkyl;-   R⁸ is OH, CN, halo, or C₁₋₄ alkoxy,-   L_(a) is absent, or is —C(═O)—, —C(═O)O—, or —C(═O)NH—;-   R^(a) is:    -   H,    -   C₁₋₄ alkyl optionally substituted with one or more independently        selected R^(b),    -   C₃₋₇ monocyclic cycloalkyl optionally substituted with one or        more independently selected R^(c), or    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S, or    -   5-6 membered monocyclic heteroaryl comprising one or more        heteroatoms independently selected from O, N, and S;-   R^(b) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkoxy,    -   C₃₋₇ cycloallkyl,    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S (which        heterocycloalkyl is optionally substituted with one or more        independently selected halo, or oxo),    -   —SO₂—C₁₋₄ alkyl, or    -   —C(═O)NR^(b1)R^(b2)-   R^(c) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkyl,    -   —C(═O)OH, or    -   —C(═O)NR^(c1)R^(c2); and-   each R^(b1), R^(b2), R^(c1) and R^(c2) is independently selected    from H, and C₁₋₄ alkyl.

In a particular embodiment the compounds of the invention are inhibitorsof JAK1 and/or TYK2.

Surprisingly, it has now been found that the compounds of the inventionmay exhibit improved in vitro activity when compared to closely relatedanalogues.

Furthermore, in a particular aspect, the compounds of the invention mayexhibit improved stability in vitro, when compared to closely relatedanalogues. This improvement may result in vivo in a lower dosage of thedrug being required, and thereby may result in decreased toxicity and/ordrug-drug interaction.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising the compounds of the invention, and apharmaceutical carrier, excipient or diluent. Moreover, the compounds ofthe invention, useful in the pharmaceutical compositions and treatmentmethods disclosed herein, are pharmaceutically acceptable as preparedand used. In this aspect of the invention, the pharmaceuticalcomposition may additionally comprise further active ingredientssuitable for use in combination with the compounds of the invention.

In a further aspect, the invention provides a compound of the inventionor a pharmaceutical composition comprising a compound of the inventionfor use as a medicament. In a specific embodiment, said pharmaceuticalcomposition additionally comprises a further active ingredient.

In a further aspect of the invention, this invention provides a methodof treating a mammal susceptible to or afflicted with a condition fromamong those listed herein, and particularly, such condition as may beassociated with aberrant JAK activity, e.g. allergic diseases,inflammatory diseases, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or hypersecretion of interferons, whichmethod comprises administering an effective amount of the pharmaceuticalcomposition or compound of the invention as described herein. In aspecific embodiment the condition is associated with aberrant JAK1and/or TYK2 activity.

In a further aspect, the present invention provides a compound of theinvention for use in the treatment or prophylaxis of a conditionselected from those listed herein, particularly such conditions as maybe associated with aberrant JAK activity, e.g. allergic diseases,inflammatory diseases, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or hypersecretion of interferons.

In yet another method of treatment aspect, this invention provides amethod for treating a mammal susceptible to or afflicted with acondition that is causally related to abnormal JAK activity as describedherein, and comprises administering an effective condition-treating orcondition-preventing amount of the pharmaceutical composition or acompound of the invention described herein. In a specific aspect thecondition is causally related to abnormal JAK1 and/or TYK2 activity.

In a further aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention, for use as a medicament.

In a further aspect, the present invention provides a compound of theinvention for use in the treatment or prophylaxis of a condition that iscausally related to abnormal JAK activity.

In additional aspects, this invention provides methods for synthesizingthe compounds of the invention, with representative synthetic protocolsand pathways disclosed later on herein.

Accordingly, it is a principal object of this invention to provide novelcompounds, which can modify the activity of JAK and thus prevent ortreat any conditions that may be causally related thereto. In a specificaspect the compounds of the invention modulate the activity of JAK1and/or TYK2.

It is a further object of this invention to provide compounds that cantreat or alleviate conditions or symptoms of same, such as allergicdiseases, inflammatory diseases, autoimmune diseases, proliferativediseases, transplantation rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or hypersecretion of interferons,that may be causally related to the activity of JAK, in particular JAK1and/or TYK2.

A still further object of this invention is to provide a pharmaceuticalcomposition that may be used in the treatment or prophylaxis of avariety of conditions, including the diseases associated with JAKactivity such as allergic diseases, inflammatory diseases, autoimmunediseases, proliferative diseases, transplantation rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orhypersecretion of interferons. In a specific embodiment the disease isassociated with JAK1 and/or TYK2 activity.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the terms “substituted” is to be defined as set out below. It should befurther understood that the terms “groups” and “radicals” can beconsidered interchangeable when used herein.

The articles ‘a’ and ‘an’ may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example ‘an analogue’ means one analogue or more than oneanalogue.

‘Alkyl’ means straight or branched aliphatic hydrocarbon with the numberof carbon atoms specified. Particular alkyl groups have 1 to 8 carbonatoms. More particular is lower alkyl which has 1 to 6 carbon atoms. Afurther particular group has 1 to 4 carbon atoms. Exemplary straightchained groups include methyl, ethyl n-propyl, and n-butyl. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl, propylor butyl is attached to a linear alkyl chain, exemplary branched chaingroups include isopropyl, iso-butyl, t-butyl and isoamyl.

‘Alkoxy’ refers to the group —OR²⁶ where R²⁶ is alkyl with the number ofcarbon atoms specified. Particular alkoxy groups are methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,n-hexoxy, and 1,2-dimethylbutoxy. Particular alkoxy groups are loweralkoxy, i.e. with between 1 and 6 carbon atoms. Further particularalkoxy groups have between 1 and 4 carbon atoms.

‘Alkenyl’ refers to monovalent olefinically (unsaturated) hydrocarbongroups with the number of carbon atoms specified. Particular alkenyl has2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms,which can be straight-chained or branched and having at least 1 andparticularly from 1 to 2 sites of olefinic unsaturation. Particularalkenyl groups include ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂),isopropenyl (—C(CH₃)═CH₂) and the like.

‘Amino’ refers to the radical —NH₂.

‘Aryl’ refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. In particular aryl refers to an aromatic ringstructure, monocyclic or fused polycyclic, with the number of ring atomsspecified. Specifically, the term includes groups that include from 6 to10 ring members. Where the aryl group is a monocyclic ring system itpreferentially contains 6 carbon atoms. Particularly aryl groups includephenyl, and naphthyl.

‘Cycloalkyl’ refers to a non-aromatic hydrocarbyl ring structure,monocyclic, fused polycyclic, bridged polycyclic, or spirocyclic, withthe number of ring atoms specified. A cycloalkyl may have from 3 to 12carbon atoms, in particular from 3 to 10, and more particularly from 3to 7 carbon atoms. Such cycloalkyl groups include, by way of example,single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cycloheptyl.

‘Cyano’ refers to the radical —CN.

‘Halo’ or ‘halogen’ refers to fluoro (F), chloro (Cl), bromo (Br) andiodo (I). Particular halo groups are either fluoro or chloro.

‘Hetero’ when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, cycloalkyl, e.g. heterocycloalkyl, aryl, e.g.heteroaryl, and the like having from 1 to 4, and particularly from 1 to3 heteroatoms, more typically 1 or 2 heteroatoms, for example a singleheteroatom.

‘Heteroaryl’ means an aromatic ring structure, monocyclic or fusedpolycyclic, that includes one or more heteroatoms independently selectedfrom O, N and S and the number of ring atoms specified. In particular,the aromatic ring structure may have from 5 to 9 ring members. Theheteroaryl group can be, for example, a five membered or six memberedmonocyclic ring or a fused bicyclic structure formed from fused five andsix membered rings or two fused six membered rings or, by way of afurther example, two fused five membered rings. Each ring may contain upto four heteroatoms typically selected from nitrogen, sulphur andoxygen. Typically the heteroaryl ring will contain up to 4 heteroatoms,more typically up to 3 heteroatoms, more usually up to 2, for example asingle heteroatom. In one embodiment, the heteroaryl ring contains atleast one ring nitrogen atom. The nitrogen atoms in the heteroaryl ringscan be basic, as in the case of an imidazole or pyridine, or essentiallynon-basic as in the case of an indole or pyrrole nitrogen. In generalthe number of basic nitrogen atoms present in the heteroaryl group,including any amino group substituents of the ring, will be less thanfive.

Examples of five membered monocyclic heteroaryl groups include but arenot limited to pyrrolyl, furanyl, thiophenyl, imidazolyl, furazanyl,oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.

Examples of six membered monocyclic heteroaryl groups include but arenot limited to pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl andtriazinyl. Particular examples of bicyclic heteroaryl groups containinga five membered ring fused to another five-membered ring include but arenot limited to imidazothiazolyl and imidazoimidazolyl. Particularexamples of bicyclic heteroaryl groups containing a six membered ringfused to a five membered ring include but are not limited tobenzfuranyl, benzthiophenyl, benzimidazolyl, benzoxazolyl,isobenzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl,isobenzofuranyl, indolyl, isoindolyl, indolizinyl, purinyl (e.g.adenine, guanine), indazolyl, pyrazolopyrimidinyl, triazolopyrimidinyl,and pyrazolopyridinyl groups. Particular examples of bicyclic heteroarylgroups containing two fused six membered rings include but are notlimited to quinolinyl, isoquinolinyl, pyridopyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinylgroups. Particular heteroaryl groups are those derived from thiophenyl,pyrrolyl, benzothiophenyl, benzofuranyl, indolyl, pyridinyl, quinolinyl,imidazolyl, oxazolyl and pyrazinyl. Examples of representativeheteroaryls include the following:

wherein each Y is selected from >C(═O), NH, O and S.

‘Heterocycloalkyl’ means a non-aromatic fully saturated ring structure,monocyclic, fused polycyclic, spirocyclic, or bridged polycyclic, thatincludes one or more heteroatoms independently selected from O, N and Sand the number of ring atoms specified. The heterocycloalkyl ringstructure may have from 4 to 12 ring members, in particular from 4 to 10ring members and more particularly from 4 to 7 ring members. Each ringmay contain up to four heteroatoms typically selected from nitrogen,sulphur and oxygen. Typically the heterocycloalkyl ring will contain upto 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to2, for example a single heteroatom. Examples of heterocyclic ringsinclude, but are not limited to azetidinyl, oxetanyl, thietanyl,pyrrolidinyl (e.g. 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl),morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl,imidazolinyl, oxazolinyl, thiazolinyl, piperidinyl (e.g. 1-piperidinyl,2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyranyl, dioxanyl,tetrahydropyranyl (e.g. 4-tetrahydro pyranyl), 2-pyrazolinyl,pyrazolidinyl, or piperazinyl.

Particular examples of monocyclic heterocycloalkyl groups are shown inthe following illustrative examples:

wherein each W and Y is independently selected from CH₂, NH, O and S.

Particular examples of fused bicyclic heterocycloalkyl groups are shownin the following illustrative examples:

wherein each W and Y is independently selected from CH₂, NH, O and S.

Particular examples of bridged bicyclic heterocycloalkyl groups areshown in the following illustrative examples:

wherein each W and Y is independently selected from CH₂, NH, O and S,and Z is N or CH.

Particular examples of spirocyclic heterocycloalkyl groups are shown inthe following illustrative examples:

wherein each Y is selected from NH, O and S.

‘Hydroxyl’ refers to the radical —OH.

‘Oxo’ refers to the radical ═O.

‘Substituted’ refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).

‘Sulfo’ or ‘sulfonic acid’ refers to a radical such as —SO₃H.

‘Thiol’ refers to the group —SH.

As used herein, term ‘substituted with one or more’ refers to one tofour substituents. In one embodiment it refers to one to threesubstituents. In further embodiments it refers to one or twosubstituents. In a yet further embodiment it refers to one substituent.

‘Thioalkoxy’ refers to the group —SR²⁶ where R²⁶ has the number ofcarbon atoms specified and particularly C₁-C₈ alkyl. Particularthioalkoxy groups are thiomethoxy, thioethoxy, n-thiopropoxy,isothiopropoxy, n-thiobutoxy, tert-thiobutoxy, sec-thiobutoxy,n-thiopentoxy, n-thiohexoxy, and 1,2-dimethylthiobutoxy. Particularthioalkoxy groups are lower thioalkoxy, i.e. with between 1 and 6 carbonatoms. Further particular alkoxy groups have between 1 and 4 carbonatoms.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable salt’ refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g. an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term‘pharmaceutically acceptable cation’ refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

‘Prodrugs’ refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding. Conventional solvents include water, EtOH,acetic acid and the like. The compounds of the invention may be preparede.g. in crystalline form and may be solvated or hydrated. Suitablesolvates include pharmaceutically acceptable solvates, such as hydrates,and further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. ‘Solvate’ encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

‘Subject’ includes humans. The terms ‘human’, ‘patient’ and ‘subject’are used interchangeably herein.

‘Effective amount’ means the amount of a compound of the invention that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “effective amount” can varydepending on the compound, the disease and its severity, and the age,weight, etc., of the subject to be treated.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringor developing a disease or disorder (i.e. causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e. arresting thedisease or reducing the manifestation, extent or severity of at leastone of the clinical symptoms thereof). In another embodiment ‘treating’or ‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or disorder,either physically, (e.g. stabilization of a discernible symptom),physiologically, (e.g. stabilization of a physical parameter), or both.In a further embodiment, “treating” or “treatment” relates to slowingthe progression of the disease.

As used herein the term ‘allergic disease(s)’ refers to the group ofconditions characterized by a hypersensitivity disorder of the immunesystem including, allergic airway disease (e.g. asthma, rhinitis),sinusitis, eczema and hives, as well as food allergies or allergies toinsect venom.

As used herein the term ‘asthma’ as used herein refers to any disorderof the lungs characterized by variations in pulmonary gas flowassociated with airway constriction of whatever cause (intrinsic,extrinsic, or both; allergic or non-allergic). The term asthma may beused with one or more adjectives to indicate the cause.

As used herein the term ‘inflammatory disease(s)’ refers to the group ofconditions including, rheumatoid arthritis, osteoarthritis, juvenileidiopathic arthritis, psoriasis, psoriatic arthritis, ankylosingspondylitis, allergic airway disease (e.g. asthma, rhinitis), chronicobstructive pulmonary disease (COPD), inflammatory bowel diseases (e.g.Crohn's disease, ulcerative colitis), endotoxin-driven disease states(e.g. complications after bypass surgery or chronic endotoxin statescontributing to e.g. chronic cardiac failure), and related diseasesinvolving cartilage, such as that of the joints. Particularly the termrefers to rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma), chronic obstructive pulmonary disease (COPD) andinflammatory bowel diseases. More particularly the term refers torheumatoid arthritis, chronic obstructive pulmonary disease (COPD) andinflammatory bowel diseases

As used herein the term ‘autoimmune disease(s)’ refers to the group ofdiseases including obstructive airways disease, including conditionssuch as COPD, asthma (e.g intrinsic asthma, extrinsic asthma, dustasthma, infantile asthma) particularly chronic or inveterate asthma (forexample late asthma and airway hyperreponsiveness), bronchitis,including bronchial asthma, systemic lupus erythematosus (SLE),cutaneous lupus erythrematosis, lupus nephritis, dermatomyositis,Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, typeI diabetes mellitus and complications associated therewith, atopiceczema (atopic dermatitis), thyroiditis (Hashimoto's and autoimmunethyroiditis), contact dermatitis and further eczematous dermatitis,inflammatory bowel disease (e.g. Crohn's disease and ulcerativecolitis), atherosclerosis and amyotrophic lateral sclerosis.Particularly the term refers to COPD, asthma, systemic lupuserythematosis, type I diabetes mellitus and inflammatory bowel disease.As used herein the term ‘proliferative disease(s)’ refers to conditionssuch as cancer (e.g. uterine leiomyosarcoma or prostate cancer),myeloproliferative disorders (e.g. polycythemia vera, essentialthrombocytosis and myelofibrosis), leukemia (e.g. acute myeloidleukaemia, acute and chronic lymphoblastic leukemia), multiple myeloma,psoriasis, restenosis, scleroderma or fibrosis. In particular the termrefers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term ‘cancer’ refers to a malignant or benign growthof cells in skin or in body organs, for example but without limitation,breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancertends to infiltrate into adjacent tissue and spread (metastasise) todistant organs, for example to bone, liver, lung or the brain. As usedherein the term cancer includes both metastatic tumour cell types (suchas but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (suchas but not limited to, colorectal cancer, prostate cancer, small celllung cancer and non-small cell lung cancer, breast cancer, pancreaticcancer, bladder cancer, renal cancer, gastric cancer, glioblastoma,primary liver cancer, ovarian cancer, prostate cancer and uterineleiomyosarcoma). In particular, the term ‘cancer’ refers to acutelymphoblastic leukemia, acute myeloidleukemia, adrenocortical carcinoma,anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, bladder cancer, bonecancer (osteosarcoma and malignant fibrous histiocytoma), brain stemglioma, brain tumors, brain and spinal cord tumors, breast cancer,bronchial tumors, Burkitt lymphoma, cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cutaneous T-Cell lymphoma, embryonal tumors,endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer,ewing sarcoma family of tumors, eye cancer, retinoblastoma, gallbladdercancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), gastrointestinal stromal celltumor, germ cell tumor, glioma, hairy cell leukemia, head and neckcancer, hepatocellular (liver) cancer, hodgkin lymphoma, hypopharyngealcancer, intraocular melanoma, islet cell tumors (endocrine pancreas),Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngealcancer, leukemia, Acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, liver cancer, non-small cell lung cancer, small cell lungcancer, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, lymphoma, Waldenstrom macroglobulinemia,medulloblastoma, medulloepithelioma, melanoma, mesothelioma, mouthcancer, chronic myelogenous leukemia, myeloid leukemia, multiplemyeloma, asopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma,non-small cell lung cancer, oral cancer, oropharyngeal cancer,osteosarcoma, malignant fibrous histiocytoma of bone, ovarian cancer,ovarian epithelial cancer, ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, papillomatosis,parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymaltumors of intermediate differentiation, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary tumor, plasma cellneoplasm/multiple myeloma, pleuropulmonary blastoma, primary centralnervous system lymphoma, prostate cancer, rectal cancer, renal cell(kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sarcoma, Ewing sarcoma family of tumors, sarcoma, kaposi, Sezarysyndrome, skin cancer, small cell Lung cancer, small intestine cancer,soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethralcancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,Waldenstrom macroglobulinemia, and Wilms tumor. In another particularembodiment, the term cancer refers to pancreatic cancer, liver cancer,hepatocellular carcinoma (HCC), breast cancer, or colon cancer.

As used herein the term ‘leukemia’ refers to neoplastic diseases of theblood and blood forming organs. Such diseases can cause bone marrow andimmune system dysfunction, which renders the host highly susceptible toinfection and bleeding. In particular the term leukemia refers to acutemyeloid leukaemia (AML), and acute lymphoblastic leukemia (ALL) andchronic lymphoblastic leukaemia (CLL). In another particular embodiment,the term leukemia refers to T-cell acute lymphoblastic leukemia (T-ALL),chronic lymphocytic leukemia (CLL), or diffuse large B-cell lymphoma(DLBCL).

As used herein the term ‘transplantation rejection’ refers to the acuteor chronic rejection of cells, tissue or solid organ allo- or xenograftsof e.g. pancreatic islets, stem cells, bone marrow, skin, muscle,corneal tissue, neuronal tissue, heart, lung, combined heart-lung,kidney, liver, bowel, pancreas, trachea or esophagus, orgraft-versus-host diseases.

As used herein the term ‘diseases involving impairment of cartilageturnover’ includes conditions such as osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,endemic forms of arthritis like osteoarthritis deformans endemica,Mseleni disease and Handigodu disease; degeneration resulting fromfibromyalgia, systemic lupus erythematosus, scleroderma and ankylosingspondylitis.

As used herein the term ‘congenital cartilage malformation(s)’ includesconditions such as hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

As used herein the term ‘disease(s) associated with hypersecretion ofIL6’ includes conditions such as Castleman's disease, multiple myeloma,psoriasis, Kaposi's sarcoma and/or mesangial proliferativeglomerulonephritis.

As used herein the term ‘disease(s) associated with hypersecretion ofinterferons includes conditions such as systemic and cutaneous lupuserythematosis, lupus nephritis, dennatomyositis, Sjogren's syndrome,psoriasis, rheumatoid arthritis.

‘Compound(s) of the invention’, and equivalent expressions, are meant toembrace compounds of the Formula(e) as herein described, whichexpression includes the pharmaceutically acceptable salts, and thesolvates, e.g. hydrates, and the solvates of the pharmaceuticallyacceptable salts where the context so permits. Similarly, reference tointermediates, whether or not they themselves are claimed, is meant toembrace their salts, and solvates, where the context so permits.

When ranges are referred to herein, for example but without limitation,C₁₋₈ alkyl, the citation of a range should be considered arepresentation of each member of said range.

Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but in the acid sensitiveform often offers advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (Bundgaard, 1985). Prodrugsinclude acid derivatives well know to practitioners of the art, such as,for example, esters prepared by reaction of the parent acid with asuitable alcohol, or amides prepared by reaction of the parent acidcompound with a substituted or unsubstituted amine, or acid anhydrides,or mixed anhydrides. Simple aliphatic or aromatic esters, amides andanhydrides derived from acidic groups pendant on the compounds of thisinvention are particularly useful prodrugs. In some cases it isdesirable to prepare double ester type prodrugs such as (acyloxy)alkylesters or ((alkoxycarbonyl)oxy)alkylesters. Particular such prodrugs arethe C₁₋₈ alkyl, C₂₋₈ alkenyl, C₆₋₁₀ optionally substituted aryl, and(C₆₋₁₀ aryl)-(C₁₋₄ alkyl) esters of the compounds of the invention.

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitro (¹⁵N), or the like.It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Further, compounds may beprepared that are substituted with positron emitting isotopes, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy.

All isotopic variants of the compounds provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed ‘isomers’. Isomersthat differ in the arrangement of their atoms in space are termed‘stereoisomers’.

Stereoisomers that are not mirror images of one another are termed‘diastereomers’ and those that are non-superimposable mirror images ofeach other are termed ‘enantiomers’. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e. as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a ‘racemic mixture’.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

The compounds of the invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)- stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

It will be appreciated that compounds of the invention may bemetabolized to yield biologically active metabolites.

The Invention

The present invention is based on the identification that the compoundsof the invention are inhibitors of JAK and that they are useful for thetreatment of allergic diseases, inflammatory diseases, autoimmunediseases, proliferative diseases, transplantation rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orhypersecretion of interferons. In a specific embodiment the compounds ofthe invention are inhibitors of JAK1 and/or TYK2.

The present invention also provides methods for the production of thecompounds of the invention, pharmaceutical compositions comprising acompound of the invention and methods for treating allergic diseases,inflammatory diseases, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or hypersecretion of interferons byadministering a compound of the invention. In a specific embodiment thecompounds of the invention are inhibitors of JAK1 and/or TYK2.

Accordingly, in a first aspect of the invention, the compounds of theinvention are provided according to Formula (I):

wherein

-   R¹ is H, or Me;-   L₁ is —NR²—; —O—, or —CH₂—;-   Cy is phenyl, or 5-9 membered monocyclic or fused bicyclic    heteroaryl comprising 1 to 4 heteroatoms independently selected from    N, O, and S;-   R² is H, or C₁₋₄ alkyl;-   R³ is H, halo, C₁₋₄ alkyl optionally substituted with one or more    independently selected halo, or C₁₋₄ alkoxy optionally substituted    with one or more independently selected halo;-   R⁴ is H, or halo;-   R⁵ is —CN, halo, or is -L₂-R⁶;-   -L₂ is absent, or is —C(═O)—, —C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂—,    —SO₂NR⁷—, or —NR⁷SO₂—;-   R⁶ is H, or C₁₋₆ alkyl optionally substituted with one or more    independently selected R groups;-   R⁷ is H, or C₁₋₄ alkyl;-   R⁸ is OH, CN, halo, or C₁₋₄ alkoxy;-   L_(a) is absent, or is —C(═O)—, —C(═O)O—, or —C(═O)NH—;-   R^(a) is:    -   H,    -   C₁₋₄ alkyl optionally substituted with one or more independently        selected R^(b),    -   C₃₋₇ monocyclic cycloalkyl optionally substituted with one or        more independently selected R^(c),    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S, or    -   5-6 membered monocyclic heteroaryl comprising one or more        heteroatoms independently selected from O, N, and S;-   R^(b) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkoxy,    -   C₃₋₇ cycloalkyl,    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S (which        heterocycloalkyl is optionally substituted with one or more        independently selected halo, or oxo),    -   —SO₂—C₁₋₄ alkyl, or    -   —C(═O)NR^(b1)R^(b2);-   R^(c) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkyl,    -   —C(═O)OH, or    -   —C(═O)NR^(c1)R^(c2); and-   each R^(b1), R^(b2), R^(c1) and R^(c2) is independently selected    from H, and C₁₋₄ alkyl.

In one embodiment, a compound of the invention is according to FormulaI, wherein R¹ is Me.

In another embodiment, a compound of the invention is according toFormula I, wherein Cy is phenyl.

In one embodiment, a compound of the invention is according to FormulaI, wherein Cy is 5-9 membered monocyclic or fused bicyclic heteroarylcomprising 1 to 4 heteroatoms independently selected from N, O, and S.In another embodiment, a compound of the invention is according toFormula I, wherein Cy is 5-9-membered monocyclic or fused bicyclicheteroaryl comprising 1 or 2 heteroatoms independently selected from N,O, and S. In a particular embodiment, Cy is pyrazolyl, pyrrolyl,imidazolyl, triazolyl, thiophenyl, thiazolyl, furanyl, pyridyl,pyrazinyl, pyrimidyl, pyridazinyl, benzofuranyl, benzothiophenyl,benzothiazolyl, benzothiadiazolyl, benzoxazolyl, indolyl or indazolyl.

In another embodiment, a compound of the invention is according toFormula I, wherein Cy is 5-6 membered monocyclic heteroaryl comprising 1to 4 heteroatoms independently selected from N, O, and S. In yet anotherembodiment, a compound of the invention is according to Formula I,wherein Cy is 5-6 membered monocyclic heteroaryl comprising 1 or 2heteroatoms independently selected from N, O, and S. In a particularembodiment, Cy is pyrazolyl, pyrrolyl, imidazolyl, furanyl, thiophenyl,triazolyl, tetrazolyl, thiazolyl, oxazolyl, thiadiazolyl, oroxadiazolyl. In another particular embodiment, Cy is pyridinyl,pyrazinyl, pyrimidinyl, or pyridazolyl. In a more particular embodiment,Cy is pyridyl.

In one embodiment, a compound of the invention is according to FormulaIIa, IIb, or IIc:

wherein L₁, R³, R⁴, L_(a), R^(a) and R⁵ are as described in any of theembodiments above.

In one embodiment, a compound of the invention is according to FormulaIIIa, IIIb, or IIIc:

wherein L₁, R³, R⁴, L_(a), R^(a) and R⁵ are as described in any of theembodiments above.

In one embodiment, a compound of the invention is according to any oneof Formulae I-IIIc, wherein L₁ is —CH₂—.

In one embodiment, a compound of the invention is according to any oneof Formulae I-IIIc, wherein L₁ is —O—.

In one embodiment, a compound of the invention is according to any oneof Formulae I-IIIc, wherein L₁ is —NR²—, and R² is as described in anyof the embodiments above.

In one embodiment, a compound of the invention is according to any oneof Formulae I-IIIc, wherein L₁ is —NR²—, wherein R² is H.

In one embodiment, a compound of the invention is according to any oneof Formulae I-IIIc, wherein L₁ is —NR²—, wherein R² is C₁₋₄ alkyl. In aparticular embodiment, R² is Me, Et, or iPr. In a more particularembodiment, R² is Me. In another more particular embodiment, R² is Et.

In one embodiment, a compound of the invention is according to FormulaIVa, IVb, IVc, IVd, IVe or IVf:

wherein R³, R⁴, R⁵, L_(a), and R^(a) are as described in any of theembodiments above.

In one embodiment, a compound of the invention is according to any oneof Formula I-IVf, wherein L_(a) is absent.

In one embodiment, a compound of the invention is according to any oneof Formula I-IVf, wherein L_(a) is —C(═O)—, —C(═O)O—, or —C(═O)NH—. In aparticular embodiment, L_(a) is —C(═O)—.

In one embodiment, a compound of the invention is according to FormulaVa, Vb, Vc, Vd, Ve or Vf:

wherein R³, R⁴, R⁵, and R^(a) are as described in any of the embodimentsabove.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R⁴ is H, or halo. In a particular embodiment,R⁴ is F, or Cl. In another particular embodiment, R⁴ is H.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R³ is H.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R³ is halo. In a particular embodiment, R³ isF, or Cl. In more particular embodiment, R³ is Cl.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R³ is C₁₋₄ alkyl. In a particular embodiment,R³ is Me, Et, or n-Pr. In more particular embodiment, R³ is Me or Et. Ina most particular embodiment, R³ is Et. In another most particularembodiment, R³ is Me.

In another embodiment, a compound of the invention is according to anyone of Formulae I-Vf, wherein R³ is C₁₋₄ alkyl substituted with one ormore independently selected halo. In a particular embodiment, R³ is—CHF₂, —CF₃, —CH₂—CHF₂ or —CH₂—CF₃. In more particular embodiment, R³ is—CF₃, or —CH₂—CF₃.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R³ is C₁₋₄ alkoxy. In a particular embodiment,R³ is —OMe, —OEt, or —On-Pr. In more particular embodiment, R³ is —OMeor —OEt.

In another embodiment, a compound of the invention is according to anyone of Formulae I-Vf, wherein R³ is C₁₋₄ alkoxy substituted with one ormore independently selected halo. In a particular embodiment, R³ is—OCHF₂, —OCF₃, or —OCH₂—CHF₂. In more particular embodiment, R³ is—OCHF₂.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is CN.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is halo. In a particular embodiment, R⁵ isF, or Cl. In a more particular embodiment, R⁵ is F.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is -L₂-R⁶, R⁶ is as described above, L₂ is—C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂NR⁷—, or —NR⁷SO₂—, and R⁷ is as defined inany of the preceding embodiments. In a particular embodiment, R⁷ is H.In another particular embodiment, R⁷ is C₁₋₄ alkyl. In a more particularembodiment, R⁷ is Me, or Et. In a most particular embodiment, R⁷ is Me.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is -L₂-R⁶, L₂ is as described above, and R⁶is H.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is -L₂-R⁶, L₂ is as described above, and R⁶is C₁₋₆ alkyl. In a particular embodiment, R⁶ is Me, Et, iPr, or tBu. Ina more particular embodiment, R⁶ is Me, or Et.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is -L₂-R⁶, L₂ is as described above, and R⁶is C₁₋₆ alkyl substituted with one or more independently selected R⁸groups. In another embodiment, R⁶ is Me, Et, iPr, or tBu, each of whichis substituted with one or more independently selected R⁸ groups. In aparticular embodiment, R⁶ is C₁₋₆ alkyl substituted with one, two orthree independently selected R⁸ groups. In another particularembodiment, R⁶ is Me, Et, iPr, or tBu, each of which is substituted withone, two or three independently selected R⁸ groups. In a more particularembodiment, R⁶ is C₁₋₆ alkyl substituted with one R⁸ group. In anotherparticular embodiment, R⁶ is Me, Et, iPr, or tBu, each of which issubstituted with one R⁸ group.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁸ is OH, CN, halo, or C₁₋₄ alkoxy. In aparticular embodiment, R⁸ is OH, CN, F, Cl, —OMe, or —OEt.

In a particular embodiment, R⁵ is -L₂-R⁶, L₂ is —C(═O)—, or —SO₂—, andR⁶ is C₁₋₆ alkyl. In a more particular embodiment, R⁵ is -L₂-R⁶, L₂ is—C(═O)—, or —SO₂, and R⁶ is Me, Et, iPr, or tBu. In a most particularembodiment, R⁵ is —C(═O)Me, —C(═O)Et, —SO₂Me or —SO₂Et.

In one embodiment, a compound of the invention is according to any oneof Formulae I-Vf, wherein R⁵ is -L₂-R⁶, L₂ is —C(═O)NR⁷—, —NR⁷C(═O)—,—SO₂NR⁷—, or —NR⁷SO₂—, R⁶ and R⁷ are as defined in any of the precedingembodiments. In a particular embodiment, R⁷ is H, Me, or Et, and R⁶ isas defined in any of the preceding embodiments. In another particularembodiment, R⁷ is as defined in any of the preceding embodiments, and R⁶is H. In yet another particular embodiment, R⁷ is as defined in any ofthe preceding embodiments, and R⁶ is C₁₋₆ alkyl optionally substitutedwith one or more independently selected OH, CN, halo, or C₁₋₄ alkoxy. Ina more particular embodiment, R⁷ is H, Me, or Et, and R⁶ is H. Inanother more particular embodiment, R⁷ is H, Me, or Et, and R⁶ is Me,Et, iPr or tBu, each of which is optionally substituted with one or moreindependently selected OH, CN, halo, or C₁₋₄ alkoxy. In yet another moreparticular embodiment, R⁷ is H, Me, or Et, and R⁶ is Me, Et, iPr or tBu,each of which is optionally substituted with one or more independentlyselected OH, CN, F, Cl, OMe, or OEt. In a most particular embodiment, R⁵is —C(═O)NH₂, or —SO₂NH₂.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is H.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is C₁₋₄ alkyl. In a particularembodiment, R^(a) is Me, Et, iPr, or tBu. In a more particularembodiment, R^(a) is Me, or Et. In a most particular embodiment, R^(a)is Me.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is C₁₋₄ alkyl substituted with one ormore independently selected R^(b). In another embodiment, R^(a) is Me,Et, iPr, or tBu, each of which is substituted with one or moreindependently selected R^(b). In a particular embodiment, R^(a) is C₁₋₄alkyl substituted with one, two, or three independently selected R^(b).In another particular embodiment, R^(a) is Me, Et, iPr, or tBu, each ofwhich is substituted with one, two, or three independently selectedR^(b). In a more particular embodiment, R^(a) is C₁₋₄ alkyl substitutedwith one R^(b). In another more particular embodiment, R^(a) is Me, Et,iPr, or tBu, each of which is substituted with one R^(b).

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is halo, CN, or OH. In a particularembodiment, R^(b) is F, Cl, CN, or OH.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is C₁₋₄ alkoxy. In a particularembodiment, R^(b) is OMe, OEt, or OiPr. In a more particular embodiment,R^(b) is OMe.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is C₃₋₇ cycloalkyl. In a particularembodiment, R^(b) is cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl. In a more particular embodiment, R^(b) is cyclopropyl.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is 4-7 membered monocyclicheterocycloalkyl comprising one or more heteroatoms independentlyselected from O, N, and S. In a particular embodiment, R^(b) isoxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiomorpholinyl.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is 4-7 membered monocyclicheterocycloalkyl comprising one or more heteroatoms independentlyselected from O, N, and S, substituted with one or more independentlyselected halo, or oxo. In a particular embodiment, R^(b) is oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, or thiomorpholinyl, each of whichis substituted with one or more independently selected halo, or oxo. Inanother particular embodiment, R^(b) is 4-7 membered monocyclicheterocycloalkyl comprising one or more heteroatoms independentlyselected from O, N, and S, substituted with one or more independentlyselected F, Cl, or oxo. In a more particular embodiment, R^(b) isoxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiomorpholinyl,each of which is substituted with one or more independently selected F,Cl, or oxo. In a most particular embodiment, R^(b) is 4-7 memberedmonocyclic heterocycloalkyl comprising one or more heteroatomsindependently selected from O, N, and S, substituted with one, two orthree independently selected F, Cl, or oxo. In another most particularembodiment, R^(b) is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, orthiomorpholinyl, each of which is substituted with one, two or threeindependently selected F, Cl, or oxo.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is —SO₂—C₁₋₄ alkyl. In a particularembodiment, R^(b) is —SO₂CH₃, or —SO₂CH₂CH₃.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(b) is —C(═O)NR^(b1)R^(b2), wherein eachR^(b1), or R^(b2) is independently selected from H, and C₁₋₄ alkyl. In aparticular embodiment, each R^(b1), or R^(b2) is independently selectedfrom H, —CH₃, and —CH₂CH₃. In a more particular embodiment, R^(b) is—C(═O)NH₂, —C(═O)NMeH, or —C(═O)NMe₂.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is C₃₋₇ monocyclic cycloalkyl. In aparticular embodiment, R^(a) is cyclopropyl, cyclobutyl, or cyclopentyl.In a more particular embodiment, R^(a) is cyclopropyl.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is C₃₋₇ monocyclic cycloalkyl substitutedwith one or more independently selected R^(c) groups. In anotherembodiment, R^(a) is cyclopropyl, cyclobutyl, or cyclopentyl, each ofwhich is substituted with one or more independently selected R^(c)groups. In a particular embodiment, R^(a) is C₃₋₇ monocyclic cycloalkylsubstituted with one, two or three independently selected R^(c) groups.In another particular embodiment, R^(a) is cyclopropyl, cyclobutyl, orcyclopentyl, each of which is substituted with one, two or threeindependently selected R^(c) groups. In a more particular embodiment,R^(a) is C₃₋₇ monocyclic cycloalkyl substituted with one R^(c) group. Inanother particular embodiment, R^(a) is cyclopropyl, cyclobutyl, orcyclopentyl, each of which is substituted with one R^(c) group.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(c) is halo, CN, or OH. In a particularembodiment, R^(c) is F, Cl, CN, or OH. In a more particular embodiment,R^(c) is F.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(c) is C₁₋₄ alkyl. In a particularembodiment, R^(c) is -Me, -Et, or -iPr.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(c) is —C(═O)OH.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(c) is —C(═O)NR^(c1)R^(c2) wherein eachR^(c1), or R^(c2) is independently selected from H, and C₁₋₄ alkyl. In aparticular embodiment, each R^(c1), or R^(c2) is independently selectedfrom H, —CH₃, and —CH₂CH₃. In a more particular embodiment, R^(c) is—C(═O)NH₂, —C(═O)NMeH, or —C(═O)NMe₂.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is:

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is:

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is 4-7 membered monocyclicheterocycloalkyl comprising one or more heteroatoms independentlyselected from O, N, and S. In a particular embodiment, R^(a) isoxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiomorpholinyl.

In one embodiment, a compound of the invention is according to any oneof Formula I-Vf, wherein R^(a) is 5-6 membered monocyclic heteroarylcomprising one or more heteroatoms independently selected from O, N, andS. In a particular embodiment, R^(a) is furanyl, thienyl, pyrrolyl,oxazolyl, thiazolyl, imidazolyl, triazolyl, oxadiazolyl, tetrazole,pyridinyl, pyrazinyl, or pyrimidinyl.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound is selected from:

-   N-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide,-   N-(6-(4-cyano-2-ethyl-6-fluorophenylamino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide,-   N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide,-   methyl    6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate,-   methyl    6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate,-   (1R,2R)-N-[6-[(6-cyano-4-ethyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide    (1S,2S)/(1R,2R) racemic mixture,-   (1R,2R)-N-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide,-   (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide,-   (1R,2R)-N-[6-(4-cyano-2-ethyl-phenoxy)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanccarboxamide,-   (1R,2R)-N-[6-(2-chloro-4-cyano-6-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-2-fluoro-N-[6-[(6-fluoro-4-methyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   (1R,2R)-N-[6-[(2,6-difluoro-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-N-[6-[(6-cyano-2-fluoro-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-N-[6-(4-cyano-2-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-2-fluoro-N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   (1R,2R)-2-fluoro-N-[6-(2-fluoro-N-methyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   N-[6-[(4-ethyl-6-methylsulfonyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-N-[6-[(6-cyano-2-fluoro-4-methyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-N-[6-[(2-cyano-3-fluoro-5-methyl-4-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   5-((4-(cyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino)-4-ethylpicolinamide,-   4-ethyl-5-((4-((1R,2R)-2-fluorocyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino)picolinamide,-   (1R,2R)-N-[6-(N,2-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   (1R,2R)-N-[6-(4-ethylsulfonyl-N,2-dimethyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,-   5-(7-amino-3-methyl-benzimidazol-5-yl)oxy-4-methyl-pyridine-2-carbonitrile,-   N-[6-[(4-ethyl-6-methylsulfonyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   N-[6-[4-(cyanomethyl)anilino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,-   N-[6-(2,3-dihydro-1,4-benzodioxin-6-ylamino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,    and-   5-[7-[[(1R,2R)-2-fluorocyclopropanecarbonyl]amino]-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carboxamide.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound is selected from

-   1-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl-urea,-   4-methyl-5-[3-methyl-7-(methylamino)benzimidazol-5-yl]oxy-pyridine-2-carbonitrile,-   5-[7-(dimethylamino)-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carbonitrile,-   N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-hydroxy-azetidine-1-carboxamide,-   N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]morpholine-4-carboxamide,    and-   1-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl-urea.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound isN-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.In a more particular embodiment, a compound of the invention isaccording to Formula I, wherein the compound is(1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound is notN-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.In a more particular embodiment, a compound of the invention isaccording to Formula I, wherein the compound is not(1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound isN-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.In a more particular embodiment, a compound of the invention isaccording to Formula I, wherein the compound is(1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to FormulaI, wherein the compound is notN-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.In a more particular embodiment, a compound of the invention isaccording to Formula I, wherein the compound is not(1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.

In one embodiment a compound of the invention is not an isotopicvariant.

In one aspect a compound of the invention according to any one of theembodiments herein described is present as the free base.

In one aspect a compound of the invention according to any one of theembodiments herein described is a pharmaceutically acceptable salt.

In one aspect a compound of the invention according to any one of theembodiments herein described is a solvate of the compound.

In one aspect a compound of the invention according to any one of theembodiments herein described is a solvate of a pharmaceuticallyacceptable salt of a compound.

While specified groups for each embodiment have generally been listedabove separately, a compound of the invention includes one in whichseveral or each embodiment in the above Formula, as well as otherformulae presented herein, is selected from one or more of particularmembers or groups designated respectively, for each variable. Therefore,this invention is intended to include all combinations of suchembodiments within its scope.

While specified groups for each embodiment have generally been listedabove separately, a compound of the invention may be one for which oneor more variables (for example, R groups) is selected from one or moreembodiments according to any of the Formula(e) listed above. Therefore,the present invention is intended to include all combinations ofvariables from any of the disclosed embodiments within its scope.

Alternatively, the exclusion of one or more of the specified variablesfrom a group or an embodiment, or combinations thereof is alsocontemplated by the present invention.

In certain aspects, the present invention provides prodrugs andderivatives of the compounds according to the formulae above. Prodrugsare derivatives of the compounds of the invention, which havemetabolically cleavable groups and become by solvolysis or underphysiological conditions the compounds of the invention, which arepharmaceutically active, in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters, and the like.

Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but the acid sensitive formoften offers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (Bundgard, H, 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare preferred prodrugs. In some cases it is desirable to prepare doubleester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the C₁ to C₈alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl, and C₇-C₁₂arylalkyl esters of the compounds of the invention.

Clauses

-   1) A compound according to Formula I:

wherein

-   R¹ is H, or Me;-   L₁ is —NR²—; —O—, or —CH₂—;-   Cy is phenyl, or 5-9 membered monocyclic or fused bicyclic    heteroaryl comprising 1 to 4 heteroatoms independently selected from    N, O, and S;-   R² is H, or C₁₋₄ alkyl;-   R³ is H, halo, C₁₋₄ alkyl optionally substituted with one or more    independently selected halo, or C₁₋₄ alkoxy optionally substituted    with one or more independently selected halo;-   R⁴ is H, or halo;-   R⁵ is —CN, halo, or is -L₂-R⁶;-   -L₂ is absent, or is —C(═O)—, —C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂—,    —SO₂NR⁷—, or —NR⁷SO₂—;-   R⁶ is H, or C₁₋₆ alkyl optionally substituted with one or more    independently selected R⁸ groups;-   R⁷ is H, or C₁₋₄ alkyl;-   R⁸ is OH, CN, halo, or C₁₋₄ alkoxy;-   L_(a) is absent, or is —C(═O)—, —C(═O)O—, or —C(═O)NH—;-   R^(a) is:    -   H,    -   C₁₋₄ alkyl optionally substituted with one or more independently        selected R^(b),    -   C₃₋₇ monocyclic cycloalkyl optionally substituted with one or        more independently selected R^(c),    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S, or    -   5-6 membered monocyclic heteroaryl comprising one or more        heteroatoms independently selected from O, N, and S;-   R^(b) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkoxy,    -   C₃₋₇ cycloalkyl,    -   4-7 membered monocyclic heterocycloalkyl comprising one or more        heteroatoms independently selected from O, N, and S (which        heterocycloalkyl is optionally substituted with one or more        independently selected halo, or oxo),    -   —SO₂—C₁₋₄ alkyl, or    -   —C(═O)NR^(b1)R^(b2).-   R^(c) is    -   halo,    -   CN,    -   OH,    -   C₁₋₄ alkyl,    -   —C(═O)OH, or    -   —C(═O)NR^(c1)R^(c2); and-   each R^(b1), R^(b2), R^(c1) and R^(c2) is independently selected    from H, and C₁₋₄ alkyl; or a pharmaceutically acceptable salt, or a    solvate, or a solvate of the pharmaceutically acceptable salts.-   2) A compound or pharmaceutically acceptable salt according to    clause 1, wherein R¹ is Me.-   3) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-2, wherein Cy is phenyl.-   4) A compound or pharmaceutically acceptable salt thereof according    to any one of clauses 1-2, wherein Cy is 5-9 membered monocyclic or    fused bicyclic heteroaryl comprising 1 to 4 heteroatoms    independently selected from N, O, and S.-   5) A compound or pharmaceutically acceptable salt thereof according    to any one of clauses 1-2, wherein 5-6 membered monocyclic    heteroaryl comprising 1 to 4 heteroatoms independently selected from    N, O, and S.-   6) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-2, wherein Cy is pyridyl.-   7) A compound or pharmaceutically acceptable salt according to any    one of clauses 1, wherein the compound is according to Formula IIa,    IIb, or IIc:

-    wherein L₁, R³, R⁴, L_(a), R^(a) and R⁵ are as described in clause    1.-   8) A compound or pharmaceutically acceptable salt according to any    one of clauses 1, wherein the compound is according to Formula IIIa,    IIIb, or IIIc:

-    wherein L₁, R³, R⁴, L_(a), R^(a) and R⁵ are as described in clause    1.-   9) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-8, wherein L₁ is —CH₂—.-   10) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-8, wherein L₁ is O.-   11) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-8, wherein L₁ is —NR²—.-   12) A compound or pharmaceutically acceptable salt according to    clause 11, wherein R² is H.-   13) A compound or pharmaceutically acceptable salt according to    clause 11, wherein R² is C₁₋₄ alkyl.-   14) A compound or pharmaceutically acceptable salt according to    clause 11, wherein R² is Me.-   15) A compound or pharmaceutically acceptable salt according to any    one of clauses 1, wherein the compound is according to Formula IVa,    IVb, IVc, IVd, IVe or IVf:

-    wherein R³, R⁴, R⁵, L_(a), and R^(a) are as described in clause 1.-   16) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-15, wherein L_(a) is absent.-   17) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-15, wherein L_(a) is —C(═O)—.-   18) A compound or pharmaceutically acceptable salt according to any    one of clauses 1, wherein the compound is according to Formula Va,    Vb, Vc, Vd, Ve or Vf:

-    wherein R³, R⁴, R⁵, L_(a), and R^(a) are as described in clause 1.-   19) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-18, wherein R⁴ is H.-   20) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-18, wherein R⁴ is F, or Cl.-   21) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-18, wherein R³ is H.-   22) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-18, wherein R³ is C₁₋₄ alkyl.-   23) A compound or pharmaceutically acceptable salt according to    clause 22, wherein R³ is Me or Et.-   24) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is CN.-   25) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is halo.-   26) A compound or pharmaceutically acceptable salt according to    clause 25, wherein R⁵ is F, or Cl.-   27) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is -L₂-R⁶, R⁶ is as described in    clause 1, and L₂ is —C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂NR⁷—, or —NR⁷SO₂—.-   28) A compound or pharmaceutically acceptable salt according to    clause 27, wherein R⁷ is H.-   29) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is -L₂-R⁶, L₂ is as described in    clause 1, and R⁶ is H.-   30) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-26, wherein R⁵ is -L₂-R⁶, L₂ is as described in    clause 1, and R⁶ is C₁₋₆ alkyl.-   31) A compound or pharmaceutically acceptable salt according to    clause 30, wherein R⁶ is Me.-   32) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is -L₂-R⁶, L₂ is as described in    clause 1, and R⁶ is C₁₋₆ alkyl substituted with one or more    independently selected R⁸ groups.-   33) A compound or pharmaceutically acceptable salt according to    clause 32, wherein R⁶ is Me, Et, iPr, or tBu, each of which is    substituted with one or more independently selected R⁸ groups.-   34) A compound or pharmaceutically acceptable salt according to    clause 33, wherein R⁸ is OH, CN, F, Cl, —OMe, or —OEt.-   35) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is -L₂-R⁶, wherein L₂ is —C(═O)—, or    —SO₂—, and R⁶ is C₁₋₆ alkyl.-   36) A compound or pharmaceutically acceptable salt according to    clause 35, wherein R⁵ is —C(═O)Me, —C(═O)Et, —SO₂Me or —SO₂Et.-   37) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-23, wherein R⁵ is -L₂-R⁶, wherein L₂ is —C(═O)NR⁷—,    —NR⁷C(═O)—, —SO₂NR⁷—, or —NR⁷SO₂—, wherein R⁷ is H, Me, or Et, and    R⁶ is Me, Et, iPr or tBu, each of which is optionally substituted    with one or more independently selected OH, CN, halo, or C₁₋₄    alkoxy.-   38) A compound or pharmaceutically acceptable salt according to    clause 37, wherein R⁵ is —C(═O)NH₂, or —SO₂NH₂.-   39) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is C₁₋₄ alkyl.-   40) A compound or pharmaceutically acceptable salt according to    clause 39, wherein R^(a) is Me, or Et.-   41) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is C₁₋₄ alkyl substituted with    one or more independently selected R^(b).-   42) A compound or pharmaceutically acceptable salt according to    clause 41, wherein R^(a) is Me, Et, iPr, or tBu.-   43) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is F, Cl, CN, or OH.-   44) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is OMe, OEt, or OiPr.-   45) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is cyclopropyl, cyclobutyl,    cyclopentyl, or cyclohexyl.-   46) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is oxetanyl, tetrahydrofuranyl,    tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl,    piperazinyl, morpholinyl, or thiomorpholinyl.-   47) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is oxetanyl, tetrahydrofuranyl,    tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl,    piperazinyl, morpholinyl, or thiomorpholinyl, each of which is    substituted with one or more independently selected halo, or oxo.-   48) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is —SO₂CH₃, or —SO₂CH₂CH₃.-   49) A compound or pharmaceutically acceptable salt according to    clause 41 or 42, wherein R^(b) is —C(═O)NH₂, —C(═O)NMeH, or    —C(═O)NMe₂.-   50) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is C₃₋₇ monocyclic cycloalkyl.-   51) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is C₃₋₇ monocyclic cycloalkyl    substituted with one or more independently selected R^(c) groups.-   52) A compound or pharmaceutically acceptable salt according to    clause 50, or 51, wherein R^(a) is cyclopropyl, cyclobutyl, or    cyclopentyl.-   53) A compound or pharmaceutically acceptable salt according to    clause 51, wherein R^(c) is F, Cl, CN, or OH.-   54) A compound or pharmaceutically acceptable salt according to    clause 51, wherein R^(c) is -Me, -Et, or -iPr.-   55) A compound or pharmaceutically acceptable salt according to    clause 51, wherein R^(c) is —C(═O)OH.-   56) A compound or pharmaceutically acceptable salt according to    clause 51, wherein R^(c) is —C(═O)NH₂, —C(═O)NMeH, or —C(═O)NMe₂.-   57) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is:

-   58) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is:

-   59) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is 4-7 membered monocyclic    heterocycloalkyl comprising one or more heteroatoms independently    selected from O, N, and S.-   60) A compound or pharmaceutically acceptable salt according to    clause 59, wherein R^(a) is oxetanyl, tetrahydrofuranyl,    tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl,    piperazinyl, morpholinyl, or thiomorpholinyl.-   61) A compound or pharmaceutically acceptable salt according to any    one of clauses 1-38, wherein R^(a) is 5-6 membered monocyclic    heteroaryl comprising one or more heteroatoms independently selected    from O, N, and S.-   62) A compound or pharmaceutically acceptable salt according to    clause 61, wherein R^(a) is furanyl, thienyl, pyrrolyl, oxazolyl,    thiazolyl, imidazolyl, triazolyl, oxadiazolyl, tetrazole, pyridinyl,    pyrazinyl, or pyrimidinyl.-   63) A compound, or pharmaceutically acceptable salt thereof,    according to clause 1 wherein the compound is selected from Table I-   64) A compound, or pharmaceutically acceptable salt thereof,    according to clause 1 wherein the compound is    (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.-   65) A compound, or pharmaceutically acceptable salt thereof,    according to clause 1 wherein the compound is not    (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.-   66) A compound, or pharmaceutically acceptable salt thereof,    according to clause 1 wherein the compound is    (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.-   67) A compound, or pharmaceutically acceptable salt thereof,    according to clause 1 wherein the compound is not    (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.-   68) A pharmaceutical composition comprising a pharmaceutically    acceptable carrier and a pharmaceutically effective amount of a    compound according to any one of clauses 1-67.-   69) The pharmaceutical composition according to clause 68 comprising    a further therapeutic agent.-   70) The compound or pharmaceutically acceptable salt thereof,    according to any one of clauses 1-67, or the pharmaceutical    composition according any one of clauses 68-69, for use in medicine.-   71) A compound according to any one of clauses 1-67, or the    pharmaceutical composition according any one of clauses 68-69, for    use in the treatment, or prophylaxis of allergic diseases,    inflammatory diseases, autoimmune diseases, proliferative diseases,    transplantation rejection, diseases involving impairment of    cartilage turnover, congenital cartilage malformations, and/or    diseases associated with hypersecretion of IL6 or hypersecretion of    interferons.-   72) A compound according to any one of clauses 1-67, or the    pharmaceutical composition according any one of clauses 68-69, for    use in the treatment, or prophylaxis of proliferative diseases.-   73) A compound according to clause 72, wherein the proliferative    diseases is selected from myelofibrosis, T-cell acute lymphoblastic    leukemia (T-ALL), multiple myeloma, chronic lymphocytic leukemia    (CLL), diffuse large B-cell lymphoma (DLBCL), pancreatic cancer,    liver cancer, hepatocellular carninoma (HCC), lung cancer, breast    cancer, and colon cancer.-   74) A method for the treatment, or prophylaxis of allergic diseases,    inflammatory diseases, autoimmune diseases, proliferative diseases,    transplantation rejection, diseases involving impairment of    cartilage turnover, congenital cartilage malformations, and/or    diseases associated with hypersecretion of IL6 or hypersecretion of    interferons, comprising administering an amount of compound    according to any one of clauses 1-67, or the pharmaceutical    composition according any one of clauses 68-69, sufficient to effect    said treatment, or prophylaxis.-   75) A method for the treatment, or prophylaxis of proliferative    diseases, comprising administering an amount of compound according    to any one of clauses 1-67, or the pharmaceutical composition    according any one of clauses 68-69, sufficient to effect said    treatment, or prophylaxis.-   76) A method of treatment according to clause 76, wherein the    proliferative diseases is selected from myelofibrosis, T-cell acute    lymphoblastic leukemia (T-ALL), multiple myeloma, chronic    lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),    pancreatic cancer, liver cancer, hepatocellular carninoma (HCC),    lung cancer, breast cancer, and colon cancer.-   77) The method according to any one of clause 75-77, wherein the    compound according to any one of clauses 1-67, or the pharmaceutical    composition according any one of clauses 68-69, is administered in    combination with a further therapeutic agent.-   78) The pharmaceutical composition according to clause 69, or the    method according to clause 78, wherein the further therapeutic agent    is an agent for the treatment, or prophylaxis of allergic diseases,    inflammatory diseases, autoimmune diseases, proliferative diseases,    transplantation rejection, diseases involving impairment of    cartilage turnover, congenital cartilage malformations, and/or    diseases associated with hypersecretion of IL6 or hypersecretion of    interferons.-   79) The pharmaceutical composition according to clause 69, or the    method according to clause 78, wherein the further therapeutic agent    is an agent for the treatment, or prophylaxis of myelofibrosis,    T-cell acute lymphoblastic leukemia (T-ALL), multiple mycloma,    chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma    (DLBCL), pancreatic cancer, liver cancer, hepatocellular carninoma    (HCC), lung cancer, breast cancer, and colon cancer.

Pharmaceutical Compositions

When employed as a pharmaceutical, a compound of the invention istypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound of theinvention according to Formula I. Generally, a compound of the inventionis administered in a pharmaceutically effective amount. The amount ofcompound of the invention actually administered will typically bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound of the invention administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

The pharmaceutical compositions of this invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intra-articular, intravenous, intramuscular, and intranasal. Dependingon the intended route of delivery, a compound of the invention ispreferably formulated as either injectable or oral compositions or assalves, as lotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term ‘unit dosage forms’ refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention according to Formula I isusually a minor component (from about 0.1 to about 50% by weight orpreferably from about 1 to about 40% by weight) with the remainder beingvarious vehicles or carriers and processing aids helpful for forming thedesired dosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or non-aqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compound of the inventionsof a similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound of the invention according toFormula I in such compositions is typically a minor component, oftenbeing from about 0.05 to 10% by weight with the remainder being theinjectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as an ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

A compound of the invention can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17^(th) edition, 1985,Mack Publishing Company, Easton, Pa., which is incorporated herein byreference.

A compound of the invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1—Tablets

A compound of the invention according to Formula I may be admixed as adry powder with a dry gelatin binder in an approximate 1:2 weight ratio.A minor amount of magnesium stearate may be added as a lubricant. Themixture may be formed into 240-270 mg tablets (80-90 mg of activecompound of the invention according to Formula I per tablet) in a tabletpress.

Formulation 2—Capsules

A compound of the invention according to Formula I may be admixed as adry powder with a starch diluent in an approximate 1:1 weight ratio. Themixture may be filled into 250 mg capsules (125 mg of active compound ofthe invention according to Formula I per capsule).

Formulation 3—Liquid

A compound of the invention according to Formula I (125 mg), may beadmixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultantmixture may be blended, passed through a No. 10 mesh U.S. sieve, andthen mixed with a previously made solution of microcrystalline celluloseand sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodiumbenzoate (10 mg), flavor, and color may be diluted with water and addedwith stirring. Sufficient water may then be added with stirring. Furthersufficient water may be then added to produce a total volume of 5 mL.

Formulation 4—Tablets

A compound of the invention according to Formula I may be admixed as adry powder with a dry gelatin binder in an approximate 1:2 weight ratio.A minor amount of magnesium stearate may be added as a lubricant. Themixture may be formed into 450-900 mg tablets (150-300 mg of activecompound of the invention according to Formula I) in a tablet press.

Formulation 5—Injection

A compound of the invention according to Formula I may be dissolved orsuspended in a buffered sterile saline injectable aqueous medium to aconcentration of approximately 5 mg/mL.

Formulation 6—Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted atabout 75° C. and then a mixture of A compound of the invention accordingto Formula I (50 g) methylparaben (0.25 g), propylparaben (0.15 g),sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved inwater (about 370 g) may be added and the resulting mixture may bestirred until it congeals.

Methods of Treatment

A compound of the invention may be used as a therapeutic agent for thetreatment of conditions in mammals that are causally related orattributable to aberrant activity of JAK. In particular, conditionsrelated to aberrant activity of JAK1 and/or TYK2. Accordingly, thecompounds and pharmaceutical compositions of the invention find use astherapeutics for preventing and/or treating allergic diseases,inflammatory diseases, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or hypersecretion of interferons in mammalsincluding humans.

In one aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use as a medicament.

In another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament.

In yet another aspect, the present invention provides a method oftreating a mammal having, or at risk of having a disease disclosedherein, said method comprising administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or compounds of the invention hereindescribed. In a particular aspect, the present invention provides amethod of treating a mammal having, or at risk of having allergicdiseases, inflammatory diseases, autoimmune diseases, proliferativediseases, transplantation rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or hypersecretion of interferons.

In a method of treatment aspects, this invention provides methods oftreatment and/or prophylaxis of a mammal susceptible to or afflictedwith an allergic reaction, said method comprising administering aneffective condition-treating or condition-preventing amount of one ormore of the pharmaceutical compositions or compounds of the invention asherein described. In a specific embodiment, the allergic reaction isselected from allergic airway disease, sinusitis, eczema and hives, foodallergies and allergies to insect venom.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of an allergicreaction. In a specific embodiment, the allergic reaction is selectedfrom allergic airway disease, sinusitis, eczema and hives, foodallergies and allergies to insect venom.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,or prophylaxis of an allergic reaction. In a specific embodiment, theallergic reaction is selected from allergic airway disease, sinusitis,eczema and hives, food allergies and allergies to insect venom.

In additional method of treatment aspects, this invention providesmethods of treatment and/or prophylaxis of a mammal susceptible to orafflicted with an inflammatory condition. The methods compriseadministering an effective condition-treating or condition-preventingamount of one or more of the pharmaceutical compositions or compounds ofthe invention as herein described. In a specific embodiment, theinflammatory condition is selected from rheumatoid arthritis,osteoarthritis, allergic airway disease (e.g. asthma) and inflammatorybowel diseases.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of aninflammatory condition. In a specific embodiment, the inflammatorycondition is selected from rheumatoid arthritis, osteoarthritis,allergic airway disease (e.g. asthma) and inflammatory bowel diseases.

In yet another aspect, the present invention provides the compound ofthe invention, or a pharmaceutical composition comprising a compound ofthe invention for use in the manufacture of a medicament for thetreatment, and/or prophylaxis of an inflammatory condition. In aspecific embodiment, the inflammatory condition is selected fromrheumatoid arthritis, osteoarthritis, allergic airway disease (e.g.asthma) and inflammatory bowel diseases.

In additional method of treatment aspects, this invention providesmethods of treatment and/or prophylaxis of a mammal susceptible to orafflicted with an autoimmune disease. The methods comprise administeringan effective condition-treating or condition-preventing amount of one ormore of the pharmaceutical compositions or compounds of the inventionherein described. In a specific embodiment, the autoimmune disease isselected from COPD, asthma, systemic lupus erythematosis, type Idiabetes mellitus and inflammatory bowel disease.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of an autoimmunedisease. In a specific embodiment, the autoimmune disease is selectedfrom COPD, asthma, systemic lupus erythematosis, type I diabetesmellitus and inflammatory bowel disease. In a more specific embodiment,the autoimmune disease is systemic lupus erythematosis.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of an autoimmune disease. In a specific embodiment,the autoimmune disease is selected from COPD, asthma, systemic lupuserythematosis, type I diabetes mellitus and inflammatory bowel disease.

In further method of treatment aspects, this invention provides methodsof treatment and/or prophylaxis of a mammal susceptible to or afflictedwith a proliferative disease, said methods comprising administering aneffective condition-treating or condition-preventing amount of one ormore of the pharmaceutical compositions or compounds of the inventionherein described. In a specific embodiment, the proliferative disease isselected from cancer (e.g. solid tumors such as uterine leiomyosarcomaor prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myelomaand psoriasis. In a more specific embodiment, the proliferative diseaseis selected from myelofibrosis, T-cell acute lymphoblastic leukemia(T-ALL), multiple myeloma, chronic lymphocytic leukemia (CLL), diffuselarge B-cell lymphoma (DLBCL), pancreatic cancer, liver cancer,hepatocellular carninoma (HCC), lung cancer, breast cancer, and coloncancer.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of aproliferative disease. In a specific embodiment, the proliferativedisease is selected from cancer (e.g. solid tumors such as uterineleiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL),multiple myeloma and psoriasis. In a more specific embodiment, theproliferative disease is selected from myelofibrosis, T-cell acutelymphoblastic leukemia (T-ALL), multiple mycloma, chronic lymphocyticleukemia (CLL), diffuse large B-cell lymphoma (DLBCL), pancreaticcancer, liver cancer, hepatocellular carninoma (HCC), lung cancer,breast cancer, and colon cancer.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of a proliferative disease. In a specific embodiment,the proliferative disease is selected from cancer (e.g. solid tumorssuch as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML,ALL or CLL), multiple myeloma and psoriasis. In a more specificembodiment, the proliferative disease is selected from myelofibrosis,T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),pancreatic cancer, liver cancer, hepatocellular carninoma (HCC), lungcancer, breast cancer, and colon cancer.

In further method of treatment aspects, this invention provides methodsof treatment and/or prophylaxis of a mammal susceptible to or afflictedwith transplantation rejection, said methods comprising administering aneffective condition-treating or condition-preventing amount of one ormore of the pharmaceutical compositions or compounds of the inventionherein described. In a specific embodiment, the transplantationrejection is organ transplant rejection.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis oftransplantation rejection. In a specific embodiment, the transplantationrejection is organ transplant rejection.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatmentand/or prophylaxis of of transplantation rejection. In a specificembodiment, the transplantation rejection is organ transplant rejection.

In a method of treatment aspect, this invention provides a method oftreatment, and/or prophylaxis in a mammal susceptible to or afflictedwith diseases involving impairment of cartilage turnover, which methodcomprises administering a therapeutically effective amount of a compoundof the invention, or one or more of the pharmaceutical compositionsherein described.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of diseasesinvolving impairment of cartilage turnover.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of diseases involving impairment of cartilageturnover.

The present invention also provides a method of treatment and/orprophylaxis of congenital cartilage malformations, which methodcomprises administering an effective amount of one or more of thepharmaceutical compositions or compounds of the invention hereindescribed.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of congenitalcartilage malformations.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of congenital cartilage malformations.

In further method of treatment aspects, this invention provides methodsof treatment and/or prophylaxis of a mammal susceptible to or afflictedwith diseases associated with hypersecretion of IL6, said methodscomprising administering an effective condition-treating orcondition-preventing amount of one or more of the pharmaceuticalcompositions or compounds of the invention herein described. In aspecific embodiment, the disease associated with hypersecretion of IL6is selected from Castleman's disease and mesangial proliferativeglomerulonephritis.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of diseasesassociated with hypersecretion of IL6. In a specific embodiment, thedisease associated with hypersecretion of IL6 is selected fromCastleman's disease and mesangial proliferative glomerulonephritis.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of diseases associated with hypersecretion of IL6. Ina specific embodiment, the disease associated with hypersecretion of IL6is selected from Castleman's disease and mesangial proliferativeglomerulonephritis.

In further method of treatment aspects, this invention provides methodsof treatment and/or prophylaxis of a mammal susceptible to or afflictedwith diseases associated with hypersecretion of interferons, saidmethods comprising administering an effective condition-treating orcondition-preventing amount of one or more of the pharmaceuticalcompositions or compounds of the invention herein described. In aspecific embodiment, the disease associated with hypersecretion ofinterferons is selected from systemic and cutaneous lupus erythematosis,lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, andrheumatoid arthritis.

In another aspect the present invention provides a compound of theinvention for use in the treatment, and/or prophylaxis of diseasesassociated with hypersecretion of interferons. In a specific embodiment,the disease associated with hypersecretion of interferons is selectedfrom systemic and cutaneous lupus erythematosis, lupus nephritis,dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoidarthritis.

In yet another aspect, the present invention provides a compound of theinvention, or a pharmaceutical composition comprising a compound of theinvention for use in the manufacture of a medicament for the treatment,and/or prophylaxis of diseases associated with hypersecretion ofinterferons. In a specific embodiment, the disease associated withhypersecretion of interferons is selected from systemic and cutaneouslupus erythematosis, lupus nephritis, dermatomyositis, Sjogren'ssyndrome, psoriasis, and rheumatoid arthritis.

As a further aspect of the invention there is provided a compound of theinvention for use as a pharmaceutical especially in the treatment and/orprophylaxis of the aforementioned conditions and diseases. Also providedherein is the use of the present compounds in the manufacture of amedicament for the treatment and/or prophylaxis of one of theaforementioned conditions and diseases.

A particular regimen of the present method comprises the administrationto a subject suffering from a disease involving inflammation, of aneffective amount of a compound of the invention for a period of timesufficient to reduce the level of inflammation in the subject, andpreferably terminate the processes responsible for said inflammation. Aspecial embodiment of the method comprises administering of an effectiveamount of a compound of the invention to a subject patient sufferingfrom or susceptible to the development of rheumatoid arthritis, for aperiod of time sufficient to reduce or prevent, respectively,inflammation in the joints of said patient, and preferably terminate,the processes responsible for said inflammation.

A further particular regimen of the present method comprises theadministration to a subject suffering from a disease conditioncharacterized by cartilage or joint degradation (e.g. rheumatoidarthritis and/or osteoarthritis) of an effective amount of a compound ofthe invention for a period of time sufficient to reduce and preferablyterminate the self-perpetuating processes responsible for saiddegradation. A particular embodiment of the method comprisesadministering of an effective amount of a compound of the invention to asubject patient suffering from or susceptible to the development ofosteoarthritis, for a period of time sufficient to reduce or prevent,respectively, cartilage degradation in the joints of said patient, andpreferably terminate, the self-perpetuating processes responsible forsaid degradation. In a particular embodiment said compound may exhibitcartilage anabolic and/or anti-catabolic properties.

Injection dose levels range from about 0.1 mg/kg/h to at least 10mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h.A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more mayalso be administered to achieve adequate steady state levels. Themaximum total dose is not expected to exceed about 2 g/day for a 40 to80 kg human patient.

For the prophylaxis and/or treatment of long-term conditions, such asdegenerative conditions, the regimen for treatment usually stretchesover many months or years so oral dosing is preferred for patientconvenience and tolerance. With oral dosing, one to five and especiallytwo to four and typically three oral doses per day are representativeregimens. Using these dosing patterns, each dose provides from about0.01 to about 20 mg/kg of a compound of the invention, with particulardoses each providing from about 0.1 to about 10 mg/kg and especiallyabout 1 to about 5 mg/kg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of a condition, a compound of theinvention will be administered to a patient at risk for developing thecondition, typically on the advice and under the supervision of aphysician, at the dosage levels described above. Patients at risk fordeveloping a particular condition generally include those that have afamily history of the condition, or those who have been identified bygenetic testing or screening to be particularly susceptible todeveloping the condition.

A compound of the invention can be administered as the sole active agentor it can be administered in combination with other therapeutic agents,including other compounds that demonstrate the same or a similartherapeutic activity and that are determined to be safe and efficaciousfor such combined administration. In a specific embodiment,co-administration of two (or more) agents allows for significantly lowerdoses of each to be used, thereby reducing the side effects seen.

In one embodiment, a compound of the invention or a pharmaceuticalcomposition comprising a compound of the invention is administered as amedicament. In a specific embodiment, said pharmaceutical compositionadditionally comprises a further active ingredient.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of adisease involving inflammation; particular agents include, but are notlimited to, immunoregulatory agents e.g. azathioprine, corticosteroids(e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A,tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OKT3, e.g.Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, andpiroxicam.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofarthritis (e.g. rheumatoid arthritis); particular agents include but arenot limited to analgesics, non-steroidal anti-inflanmmatory drugs(NSAIDS), steroids, synthetic DMARDS (for example but without limitationmethotrexate, leflunomide, sulfasalazine, auranofin, sodiumaurothiomalate, penicillamine, chloroquine, hydroxychloroquine,azathioprine, and ciclosporin), and biological DMARDS (for example butwithout limitation Infliximab, Etanercept, Adalimumab, Rituximab, andAbatacept).

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofproliferative disorders; particular agents include but are not limitedto: methotrcxate, lcukovorin, adriamycin, prenisone, bleomycin,cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrolacetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors(e.g. Iressa®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™),proteasome inhibitors (e.g. Velcade™), Glivec® and hsp90 inhibitors(e.g. 17-AAG). Additionally, a compound of the invention may beadministered in combination with other therapies including, but notlimited to, radiotherapy or surgery. In a specific embodiment theproliferative disorder is selected from cancer, myeloproliferativedisease and leukaemia.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofautoimmune diseases, particular agents include but are not limited to:glucocorticoids, cytostatic agents (e.g. purine analogs), alkylatingagents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas,platinum compounds, and others), antimetabolites (e.g. methotrexate,azathioprine and mercaptopurine), cytotoxic antibiotics (e.g.dactinomycin anthracyclines, mitomycin C, bleomycin, and mithramycin),antibodies (e.g. anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonalantibodies, Atgam® and Thymoglobuline®), cyclosporin, tacrolimus,rapamycin (sirolimus), interferons (e.g. IFN-β), TNF binding proteins(e.g. infliximab (Remicade™), etanercept (Enbrel™), or adalimumab(Humira™)), mycophenolate, Fingolimod and Myriocin.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis oftransplantation rejection, particular agents include but are not limitedto: calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)),mTOR inhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g.azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone,hydrocortisone), Antibodies (e.g. monoclonal anti-IL-2Rα receptorantibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies(e.g. anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of asthmaand/or rhinitis and/or COPD, particular agents include but are notlimited to: beta2-adrenoceptor agonists (e.g. salbutamol, levalbuterol,terbutaline and bitolterol), epinephrine (inhaled or tablets),anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral orinhaled) Long-acting β2-agonists (e.g. salmeterol, formoterol,bambuterol, and sustained-release oral albuterol), combinations ofinhaled steroids and long-acting bronchodilators (e.g.fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonistsand synthesis inhibitors (e.g. montelukast, zafirlukast and zileuton),inhibitors of mediator release (e.g. cromoglycate and ketotifen),biological regulators of IgE response (e.g. omalizumab), antihistamines(e.g. ceterizine, cinnarizine, fexofenadine) and vasoconstrictors (e.g.oxymethazoline, xylomethazoline, nafazoline and tramazoline).

Additionally, a compound of the invention may be administered incombination with emergency therapies for asthma and/or COPD, suchtherapies include oxygen or heliox administration, nebulized salbutamolor terbutaline (optionally combined with an anticholinergic (e.g.ipratropium), systemic steroids (oral or intravenous, e.g. prednisone,prednisolonc, methylprednisolone, dexamethasone, or hydrocortisone),intravenous salbutamol, non-specific beta-agonists, injected or inhaled(e.g. epinephrine, isoetharine, isoproterenol, metaproterenol),anticholinergics (IV or nebulized, e.g. glycopyrrolate, atropine,ipratropium), methylxanthines (theophylline, aminophylline,bamiphylline), inhalation anesthetics that have a bronchodilatory effect(e.g. isoflurane, halothane, enflurane), ketamine and intravenousmagnesium sulfate.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofinflammatory bowel disease (IBD), particular agents include but are notlimited to: glucocorticoids (e.g. prednisone, budesonide) syntheticdisease modifying, immunomodulatory agents (e.g. methotrexate,leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurineand ciclosporin) and biological disease modifying, immunomodulatoryagents (infliximab, adalimumab, rituximab, and abatacept).

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of SLE,particular agents include but are not limited to: Disease-modifyingantirheumatic drugs (DMARDs) such as antimalarials (e.g. plaquenil,hydroxychloroquine), immunosuppressants (e.g. methotrexate andazathioprine), cyclophosphamide and mycophenolic acid; immunosuppressivedrugs and analgesics, such as nonsteroidal anti-inflammatory drugs,opiates (e.g. dextropropoxyphene and co-codamol), opioids (e.g.hydrocodone, oxycodone, MS Contin, or methadone) and the fentanylduragesic transdermal patch.

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofpsoriasis, particular agents include but are not limited to: topicaltreatments such as bath solutions, moisturizers, medicated creams andointments containing coal tar, dithranol (anthralin), corticosteroidslike desoximetasone (Topicort™), fluocinonide, vitamin D3 analogues (forexample, calcipotriol), Argan oiland retinoids (etretinate, acitretin,tazarotene), systemic treatments such as methotrexate, cyclosporine,retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolatemofetil, azathioprine, tacrolimus, fumaric acid esters or biologics suchas Amevive™, Enbrel™, Humira™, Remicade™, Raptiva™ and ustekinumab (aIL-12 and IL-23 blocker). Additionally, a compound of the invention maybe administered in combination with other therapies including, but notlimited to phototherapy, or photochemotherapy (e.g. psoralen andultraviolet A phototherapy (PUVA)).

In one embodiment, a compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofallergic reaction, particular agents include but are not limited to:antihistamines (e.g. cetirizine, diphenhydramine, fexofenadine,levocetirizine), glucocorticoids (e.g. prednisone, betamethasone,beclomethasone, dexamethasone), epinephrine, theophylline oranti-leukotrienes (e.g. montelukast or zafirlukast), anti-cholinergicsand decongestants.

By co-administration is included any means of delivering two or moretherapeutic agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation this is notessential. The agents may be administered in different formulations andat different times.

Chemical Synthetic Procedures General

The compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e. reaction temperatures, times, mole ratios of reactants,solvents, pressures, etc.) are given, other process conditions can alsobe used unless otherwise stated. Optimum reaction conditions may varywith the particular reactants or solvent used, but such conditions canbe determined by one skilled in the art by routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art (Greene, T W; Wuts, P G M; 1991).

The following methods are presented with details as to the preparationof a compound of the invention as defined hereinabove and thecomparative examples. A compound of the invention may be prepared fromknown or commercially available starting materials and reagents by oneskilled in the art of organic synthesis.

All reagents were of commercial grade and were used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents were used for reactions conducted under inertatmosphere. Reagent grade solvents were used in all other cases, unlessotherwise specified. Column chromatography was performed on silica gel60 (35-70 μm). Thin layer chromatography was carried out usingpre-coated silica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectrawere recorded on a Bruker DPX 400 NMR spectrometer (400 MHz or a BrukerAdvance 300 NMR spectrometer (300 MHz). Chemical shifts (δ) for 1H NMRspectra are reported in parts per million (ppm) relative totetramethylsilane (δ 0.00) or the appropriate residual solvent peak,i.e. CHCl₃ (δ 7.27), as internal reference. Multiplicities are given assinglet (s), doublet (d), triplet (t), quartet (q), quintuplet (quin),multiplet (m) and broad (br). Electrospray MS spectra were obtained on aWaters platform LC/MS spectrometer or with Waters Acquity H-Class UPLCcoupled to a Waters Mass detector 3100 spectrometer. Columns used:Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm ID×50 mm L, Waters AcquityUPLC BEH C18 1.7 μm, 2.1 mm ID×30 mm L, or Waters Xterra MS 5 m C18,100×4.6 mm. The methods are using either MeCN/H₂O gradients (H₂Ocontains either 0.1% TFA or 0.1% NH₃) or MeOH/H₂O gradients (H₂Ocontains 0.05% TFA). Microwave heating was performed with a BiotageInitiator.

The preparative HPLC purifications were performed with a mass-directedauto-purification system coupled with a ZQ single quadrupole massspectrometer. All HPLC purifications were performed with a gradient ofH₂O (different pHs)/MeCN. Preparative HPLC separations under basicconditions were usually carried out using a BEH XBrigde C18 (5 μm, 19×5mm) precolumn and a BEH XBrigde C18 (5 μm, 19×100 mm). Separations underacidic conditions were usually carried out using CSH Select C18 (5 μm,19×5 mm) precolumn and a CSH Select C18 (5 μm, 19×100 mm). The focusedgradient was from x % to x+25% acetonitrile in water in 5 min with acycle time of 10 min. The column flow rate was 20 mL/min. The injectionvolume ranged from 200 to 750 μL. A capillary splitter was used todivert flow after column separation to the mass spectrometer which wasdiluted by 1 mL/min of make-up flow. The make-up flow is 0.1% formicacid in methanol. All samples were purified by a Waters mass directedfraction collection.

TABLE I List of abbreviations used in the experimental section:Abbreviation Definition AcOH acetic acid ATP adenosine tri-phosphateBINAP 2,2′-bis(diphenylphosphino)- 1,1′-binaphthyl br s broad singletBrettphos 2-(dicyclohexylphosphino)3,6- dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl BSA bovine serum albumin C'ial commercially available Concconcentrated Cpd compound DCM dichloromethane DMF dimethyl formamideDMSO dimethyl sulfoxide DPPA diphenylphosphoryl azide Dsc'd fullydescribed above DTT dithiothreitol EDTA ethylenediaminetetraacetic acidEGTA ethylene glycol tetraacetic acid Et₂O diethyl ether Et₃Btriethylborane Et₃N triethylamine EtOAc ethyl acetate EtOH ethanol FACSFluorescence-Activated Cell Sorting FBS fetal bovine serum g gram h hourHEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid HPLC highpressure liquid chromatography Int intermediate iPrOH iso-Propanol mmultiplet M mass MC methyl cellulose MeCN acetonitrile MeI iodomethaneMeNH₂ methyl amine MeOH methanol mg milligram min minute mL millilitermmol millimoles MOPS 3-(N-morpholino) propanesulfonic acid MS Ms′d massspectrometry measured molecular weight Mtd method MW molecular weightNCS N-chlorosuccinimide NMP N-methylpyrrolidone NMR nuclear MagneticResonnance PBS phosphate buffered saline pBSK pBluescript phagemidPd(OAc)₂ palladium(II) acetate Pd(PPh₃)₄ tetrakis(triphenylphosphine)palladium(0) Pd₂(dba)₃ tris(dibenzylideneacetone) dipalladium(0)PdCl₂(dppf). [1,1′-bis(diphenylphosphino) DCMferrocene]dichloropalladium (II), complex with dichloromethane PDXpatient-derived xenografts PET positron emission topography PMBpara-methoxybenzyl ppm parts per million p-TsOH•H₂O para-toluenesulfonicacid monohydrate rt room temperature s singlet sat saturated SiO₂ silicaSM starting material TFA trifluoroacetic acid THF tetrahydrofuranXantphos 4,5-Bis(diphenylphosphino)- 9,9-dimethylxanthene XPhos2-dicyclohexylphosphino- 2′,4′,6′-triisopropylbiphenyl

SYNTHETIC PREPARATION OF THE COMPOUND OF THE INVENTION Example 1Synthesis of the Intermediate Compounds of the Invention 1.1. Synthesisof bis-(4-methoxy-benzyl)-amine (Int.1)

p-Anisaldehyde (411 mmol), 4-methoxybenzylamine (411 mmol) and toluene(500 mL) were combined in a round bottomed flask fitted with a condenserand a Dean-Stark trap. The reaction was refluxed for 1 h during whichwater was removed from the reaction mixture. The reaction was cooled andconcentrated. The residue was dissolved in MeOH (120 mL). The mixturewas cooled to 5° C. and NaBH₄ (205 mmol) was added in portions over 45min. The reaction was slowly heated to reflux. After 2 h at reflux, thereaction was cooled to room temperature and concentrated. The residuewas dissolved in EtOAc. The organic layer was washed (3× H₂O and brine),dried (Na₂SO₄) and concentrated to yield the desired product.

1.2. Synthesis of(6-Bromo-1-methyl-H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine(Int.2)

1.2.1. Step i: 5-Bromo-1,3-difluoro-2-nitro-benzene

A solution of 4-bromo-2,6-difluoroaniline (240 mmol) in AcOH (150 mL)was added dropwise to a suspension of NaBO₃.4 H₂O (325 mmol) in AcOH(450 mL) at 70° C. during 30 min. Another 2080 mmol of NaBO₃.4 H₂O wereadded over 5 h to the mixture. During this period the mixture wasstirred at 70° C. The mixture was poured into water and extracted withEt₂O. The organic layer was combined with another Et₂O solution obtainedfrom another reaction using the same conditions described above. Themixture was concentrated. A precipitate was formed and separated byfiltration. The filtrate was concentrated to afford the desired productafter flash column chromatography (SiO₂, petroleum ether).

1.2.2. Step ii: (5-Bromo-3-fluoro-2-nitro-phenyl)-methyl-amine

2 M MeNH₂ in THF (82 mL) was added dropwise to a solution of5-bromo-1,3-difluoro-2-nitrobenzene (164 mmol) and Cs₂CO₃ (197 mmol) inTHF (1 L). The reaction mixture was stirred at 0° C. for 1 h and then atroom temperature for 1 h. The solvent was removed under reducedpressure. The residue was partitioned between EtOAc and H₂O. The twophases were separated and the organic layer was dried (Na₂SO₄) andconcentrated to afford the desired product.

1.2.3. Step iii:5-Bromo-N,N-bis-(4-methoxy-benzyl)-N′-methyl-2-nitro-benzene-1,3-diamine

A mixture of bis-(4-methoxy-benzyl)-amine (346 mmol),5-bromo-3-fluoro-N-methyl-2-nitroaniline (297 mmol) and Et₃N (891 mmol)was stirred at 120° C. for 16 h. The mixture was then cooled. The crudewas combined with another one obtained following the same proceduredescribed above. The resulting mixture was diluted in EtOAc and washed(2×0.2 M HCl, H₂O and sat. NaHCO₃). The organic phase was dried (Na₂SO₄)and concentrated to afford the desired product.

1.2.4. Step iv:5-Bromo-N,N-bis-(4-methoxy-benzyl)-N′-methyl-benzene-1,2,3-triamine

A mixture of5-Bromo-N,N-bis-(4-methoxy-benzyl)-N′-methyl-2-nitro-benzene-1,3-diamine(170 mmol), NH₄Cl (2038 mmol) and Zn (2034 mmol) in 1:1 MeOH/THF (1 L)was stirred at room temperature for 1 h. The mixture was cooled to 0° C.and HCOOH (20 mL) was added slowly. The mixture was allowed to reachroom temperature and stirred for 1 h. The mixture was filtered andcombined with another one obtained following the procedure describedabove. The resulting mixture was concentrated. The residue was dissolvedin DCM. The organic mixture was washed (sat. NH₄Cl), dried (Na₂SO₄) andconcentrated to afford the desired product.

1.2.5. Step v:(6-Bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine

5-Bromo-N,N-bis-(4-methoxy-benzyl)-N″-methyl-benzene-1,2,3-triamine (170mmol) was dissolved in a mixture of HC(OEt)₃ (100 mol) and MeCN (500mL). The mixture was stirred at 85° C. for 0.5 h and then at roomtemperature for approximately 16 h. The mixture was combined withanother one obtained following the same procedure described above. Theresulting mixture was concentrated and the residue was purified by flashcolumn chromatography (SiO₂, 15:85 to 60:40 EtOAc/petroleum ether) toobtain the desired product.

1.3. Synthesis of5-{7-[Bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile(Int.3)

1.3.1. Step i:Bis-(4-methoxy-benzyl)-[1-methyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-benzoimidazol-4-yl]-amine

A mixture of(6-bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine(54 mmol), bis(pinacolato)diboron (81 mmol), PdCl₂(dppf).DCM (2.71 mmol)and KOAc (162.5 mmol) in DMF (150 mL) was sonicated for 5 min under astream of nitrogen. The mixture was then stirred at 110° C. in a roundbottomed flask equipped with a condenser for 1 h. The mixture wasfiltered through a celite pad and the filtrate was concentrated. Theresidue was dissolved in EtOAc and the organic layer was washed (H₂O),dried (Na₂SO₄) and concentrated to afford the desired product.

1.3.2. Step ii:5-{7-[Bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile

H₂O₂ 30% wt. in H₂O (20 mL) was added to a solution ofN,N-bis(4-methoxybenzyl)-1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-4-amine(58.5 mmol) in DMF (600 mL) and the mixture was stirred forapproximately 16 h at room temperature. The reaction was quenched with5% aqueous Na₂SO₃. The mixture was extracted with EtOAc. The organiclayer was washed (H₂O), dried (Na₂SO₄) and concentrated to afford thedesired product.

1.4. General Method: Ortho-directed Iodination

Where W can be: —N═, —CH═, —C(Cl)═; Y can be: —N═, —C(CN)═

1.4.1. Method A1

A mixture of the amino aromatic or heteroaromatic starting material (1eq), Ag₂SO₄ (1 eq) and I₂ (1 eq) in EtOH is stirred at temperaturesranging from room temperature to 50° C. for a period which can vary from1 h to approximately 16 h. The mixture is filtered, concentrated anddiluted in an organic solvent. The organic mixture undergoes aqueouswork up. The solvent is removed under reduced pressure and the residueis purified by flash column chromatography to yield the desired product.

1.4.2. Illustrative Example of Method A1: Synthesis of4-Amino-3-fluoro-5-iodo-benzonitrile (Int.4)

A mixture of 4-amino-3-fluorobenzonitrile (147 mmol), I₂ (147 mmol) andAg₂SO₄ (147 mmol) in EtOH (700 mL) was stirred at room temperature for1.5 h. The mixture was filtered and concentrated. The residue wasdissolved in EtOAc and washed (sat. Na₂S₂O₃×3). The organic layer wasdried (Na₂SO₄) and concentrated. The residue was purified by flashcolumn chromatography (SiO₂, 95:5 to 70:30 cyclohexane/EtOAc) to yieldthe desired product.

1.4.3. Method A2

A mixture of the amino aromatic or heteroaromatic starting material (1eq), Ag₂SO₄ (3 to 4 eq) and I₂ (3 to 4 eq) in EtOH is stirred at 70° C.for 16 h. More equivalents of I₂ and Ag₂SO₄ can be added to increase theconversion. The mixture is filtered and concentrated. The residue ispurified by flash column chromatography to yield the desired product.

1.5. Illustrative Example of Method A2: Synthesis of5-amino-6-fluoro-4-iodo-pyridine-2-carbonitrile (Int.5)

A mixture of Int.11 (3.62 mmol), I₂ (14.5 mmol) and Ag₂SO₄ (14.5 mmol)in EtOH (200 mL) was stirred at 70° C. for 16 h. 5 more equivalents ofI₂ and Ag₂SO₄ were added and the reaction was stirred at 70° C. for afurther 72 h. The mixture was filtered, concentrated and purified byflash column chromatography (SiO₂, 20:80 to 40:60 EtOAc/cyclohexane) toyield the desired product.

1.6. General Method: Introduction of Nitrile Group by Negishi Reaction

Where R_(A) can be aryl or heteroaryl; and X can be Cl, Br or I.

1.6.1. Method B

A mixture of aryl/heteroaryl halide (1 eq), zinc cyanide (1 to 3 eq),Pd(PPh₃)₄ (0.1 to 0.2 eq) in DMF is heated at 150° C. in a microwaveapparatus for a period ranging from 5 min to 0.5 h. The mixture isfiltered and concentrated. The residue is diluted with an organicsolvent. The organic mixture undergoes work up and the solvent isremoved under reduced pressure. The residue is triturated or purified byflash column chromatography to yield the desired product.

1.6.2. Illustrative Example of Method B: Synthesis of5-fluoro-4-methyl-pyridine-2-carbonitrile (Int.7)

A microwave tube was charged with 2-chloro-5-fluoro-4-methylpyridine(5.5 mmol), Zn(CN)₂ (16.6 mmol), Pd(PPh₃)₄ (1.1 mmol) in DMF (20 mL).The mixture was stirred at 150° C. for 5 min in a microwave reactor. Themixture was combined with other crudes obtained following the proceduredescribed above. The resulting mixture was filtered. The filtrate wasconcentrated under reduced pressure. The residue was diluted with EtOAc,washed (sat. NH₄Cl), dried (Na₂SO₄) and concentrated. The residue waspurified by flash column chromatography (SiO₂, 5:95 to 15:85EtOAc/petroleum ether) to yield the desired product.

1.7. General Method: Suzuki with Methyl Boronic Acid

Where W can be: —N═, —CH═, —C(Cl)═; and Y can be: —N═, —C(CN)═; and Xcan be —I or —Br

1.7.1. Method C

A mixture of the aromatic/heteroaromatic halide (1 eq), methyl boronicacid (1.3 to 3 eq), Pd(dppf)Cl₂.DCM (0.11 to 0.2 eq) and Cs₂CO₃ (3 to 5eq) in 1,4-dioxane is stirred at 100° C. The mixture is diluted with anorganic solvent. The organic mixture undergoes aqueous work up and thesolvent is removed under reduced pressure. The residue is purified byflash column chromatography to yield the desired product.

1.7.2. Illustrative Example of Method C: Synthesis of5-amino-6-fluoro-4-methyl-pyridine-2-carbonitrile (Int.12)

A mixture of Int.5 (3 mmol), methyl boronic acid (9.1 mmol),Pd(dppf)Cl₂.DCM (0.32 mmol) and Cs₂CO₃ (15.2 mmol) in 1,4-dioxane (8 mL)was stirred at 105° C. for 5 h. The mixture was diluted (EtOAc), washed(sat. NaHCO₃), dried (Na₂SO₄) and concentrated. The residue was purifiedby flash column chromatography (SiO₂, 10:90 to 50:50 EtOAc/petroleumether) to yield the desired product.

1.8. Synthesis of 6-bromo-2-fluoro-pyridin-3-ylamine (Int.15)

A mixture of 2-fluoropyridin-3-amine (44.6 mmol) and KOAc (44.6 mmol) inAcOH was stirred at room temperature for 1 h. The mixture was cooled to0 C and Br₂ (44.6 mmol) was added dropwise. The mixture was stirred at0° C. for 15 min. The mixture was concentrated and the residue wasdissolved in EtOAc/MeOH. The organic solution was washed (sat. NaHCO₃,sat. Na₂S₂O₃), dried (Na₂SO₄) and concentrated. The residue was purifiedby flash column chromatography (SiO₂, 100:0 to 80:20 petroleumether/EtOAc) to yield the desired product.

1.9. Synthesis of 2-bromo-6-fluoro-4-methanesulfonyl-phenylamine(Int.16)

A mixture of 2-fluoro-4-methylsulfonyl-aniline (22.76 mmol) and KOAc(22.7 mmol) in AcOH was stirred at room temperature. The mixture wascooled to 0° C. and Br₂ (22.7 mmol) was added dropwise. The mixture wasstirred for 30 min at 0° C. The mixture was concentrated and the residuewas taken up in EtOAc. The organic mixture was washed (sat. NaHCO₃, sat.Na₂S₂O₃), dried (Na₂SO₄) and concentrated to yield the desired product.

1.10. Synthesis of 4-amino-3-ethyl-5-fluoro-benzonitrile (Int.17)

A mixture of Cs₂CO₃ (46 mmol) and Pd(dppf)Cl₂.DCM (0.76 mmol) wassuspended in DMF (35 mL) and degassed with nitrogen. H₂O (400 μL), 1 MEt₃B in THF (11.5 mL) and a solution of Int.4 (7.63 mmol) in DMF (5 mL)were added to the mixture and the reaction was stirred at 60° C. for 30min. The mixture was concentrated and the residue was taken up in EtOAc,washed (sat. NaHCO₃, H₂O), dried (Na₂SO₄) and concentrated. The residuewas purified by flash column chromatography (SiO2, 100:0 to 95:5petroleum ether/EtOAc) to yield the desired product.

1.11. General Method: Conversion of NH₂ into Iodide

Where R₁₀ can be Me or Et.

1.11.1. Method D

Conc. HCl (6 eq) is added dropwise to a mixture of the aminoaromatic/heteroaromatic compound (1 eq) in H₂O at 0 C. A solution ofNaNO₂ (1.05 eq) in H₂O is added dropwise. The resulting mixture isstirred at 0 C for 15 min. A solution of KI (1.05 eq) in H₂O is addeddropwise. The mixture is stirred at 0° C. for 15 min and for 1 h at roomtemperature. The mixture is extracted with an organic solvent. Afteraqueous work up and removal of the solvent under reduced pressure, theresidue can undergo trituration or chromatography to yield the desiredproduct.

1.11.2. Illustrative Example of Method D: Synthesis of5-iodo-4-methyl-pyridine-2-carbonitrile (Int.18)

Conc. HCl (0.9 mL) was added dropwise to a mixture of Int.8 (1.88 mmol)in H₂O (10 mL) at 0° C. followed by the dropwise addition of a solutionof NaNO₂ (1.97 mmol) in H₂O (0.5 mL). The resulting mixture was stirredat 0 C for 15 min. A solution of KI (1.97 mmol) in H₂O (1 mL) was addeddropwise. The resulting mixture was stirred at 0° C. for 15 min and for1 h at room temperature. The mixture was extracted with EtOAc. Theorganic layer was washed (H₂O), dried and concentrated. The residue waspurified by flash column chromatography (SiO₂, 100:0 to 75:25cyclohexane/EtOAc) to yield the desired product.

1.12. Synthesis of 6-bromo-4-methyl-pyridin-3-ylamine (Int.20)

A mixture of NH₄Cl (14.9 mmol) and iron powder (18.4 mmol) in H₂O (5 mL)was stirred at 90° C. 2-bromo-4-methyl-5-nitropyridine (2.3 mmol) wasadded in portions. The mixture was stirred at 90° C. for 1 h and 15 min.The reaction was stopped and extracted with EtOAc. The organic layer wasdried (Na₂SO₄) and concentrated to yield the desired product.

1.13. Synthesis of 4-amino-3-chloro-5-fluoro-benzonitrile (Int.21)

A mixture of 4-amino-3-fluorobenzonitrile (184 mmol) and NCS (276 mmol)in AcOH (300 mL) was stirred at 70° C. for approximately 16 h. Themixture was concentrated. H₂O was added to the residue and the solidproduct was filtered off and washed (sat. NaHCO₃ and H₂O). To eliminateH₂O, THF was added and removed under reduced pressure to yield thedesired product.

1.14. Synthesis of 3-ethyl-4-hydroxy-benzonitrile (Int.22)

Concentrated H₂SO₄ (3.4 mL) was added dropwise to a solution of4-amino-3-ethylbenzonitrile (6.84 mmol) in H₂O (12 mL) at 0° C. Asolution of NaNO₂ (7.52 mmol) in H₂O (5 mL) was added dropwise at 0° C.to the resulting mixture. The reaction was stirred at 0° C. for 30 minand at 100° C. for 2 h. The mixture was cooled to room temperature andextracted with EtOAc. The organic layer was washed (H₂O), dried andconcentrated to yield the desired product.

1.15. Synthesis of 2-chloro-3-fluoro-pyridin-4-ylamine (Int.23)

1.15.1. Step i: tert-butyl N-(2-chloro-3-fluoro-4-pyridyl)carbamate

Diphenylphosphoryl azide (DPPA) (129 mmol) was added to a mixture of2-chloro-3-fluoro-pyridine-4-carboxylic acid (85.7 mmol), Et₃N (257mmol) in 1:1 tert-BuOH/toluene (200 mL). The mixture was heated at 110°C. for 4 h. Mixture was diluted with H₂O and extracted with DCM. Theorganic layer was dried (Na₂SO₄) and concentrated. The residue waspurified by flash column chromatography (SiO₂, 100:0 to 80:20 DCM/EtOAc)to yield the desired product tert-butylN-(2-chloro-3-fluoro-4-pyridyl)carbamate.

1.15.2. Step ii: 2-Chloro-3-fluoro-pyridin-4-ylamine

A solution of tert-butyl N-(2-chloro-3-fluoro-4-pyridyl)carbamate (20.2mmol) in 1:2 TFA/DCM (45 mL) was stirred at room temperature for 6 h.The reaction mixture was concentrated and the residue was purified byflash column chromatography (SiO₂, 100:0 to 90:10 DCM/7N NH₃ in MeOH) toyield the desired product.

1.16. General Method: Buchwald Coupling with(6-Bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine(Int.2)

Where R_(A) can be optionally substituted aryl or heteroaryl.

1.16.1. Method E1

A mixture of Int.2 (1 eq), the corresponding aniline (1.1 eq), Cs₂CO₃ (2eq) and XPhos (0.3 eq) in dry toluene is purged with an inert gas beforePd(OAc)₂ (0.1 to 0.15 eq) is added. The mixture is stirred at 110° C.for approximately 16 h. The mixture may be partitioned between anorganic solvent and an aqueous phase. The two layers are separated andthe organic layer is dried and concentrated. Alternatively the reactionmixture can be filtered and concentrated. The residue may be kept assuch or purified by chromatography to yield the desired product.

1.16.2. Illustrative Example of Method E1: Synthesis of5-{7-[Bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-ylamino}-4-ethyl-pyridine-2-carbonitrile(Int.24)

A mixture of Int.2 (0.75 mmol), Int.10 (0.79 mmol), Cs₂CO₃ (1.5 mmol)and XPhos (0.225 mmol) in dry toluene (6.3 mL) was purged with argonbefore Pd(OAc)₂ (0.075 mmol) was added. The mixture was stirred at 110°C. for approximately 16 h. The mixture was diluted with H₂O andextracted with DCM. The organic layer was washed (brine), dried (Na₂SO₄)and concentrated. The residue was purified by flash columnchromatography (SiO₂, 100:0 to 50:50 cyclohexane/EtOAc) to yield thedesired product.

1.16.3. Method E2

A mixture of Int.2 (1 eq), the corresponding aniline (1.1 eq), Cs₂CO₃ (4to 5 eq) and XPhos (0.4 eq) in dry toluene is purged with an inert gasbefore Pd(OAc)₂ (0.2 to 0.3 eq) is added. The mixture is stirred at 110°C. for a period ranging from 1 h to 24 h. The mixture may be partitionedbetween an organic solvent and an aqueous phase. The two layers areseparated and the organic layer is dried and concentrated. Alternativelythe reaction mixture can be filtered and concentrated. The residue maybe kept as such or purified by chromatography to yield the desiredproduct.

1.16.4. Illustrative Example of Method E2: Synthesis of5-{7-[Bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-ylamino}-4-methyl-pyridine-2-carbonitrile(Int.27)

A mixture of Int.2 (4 mmol), Int.8 (4.3 mmol), Cs₂CO₃ (20 mmol) andXPhos (1.6 mmol) in dry toluene (35 mL) was purged with argon for 10 minbefore Pd(OAc)₂ (1.2 mmol) was added. The mixture was stirred at 110° C.for approximately 16 h. The mixture was filtered through a celite padand concentrated. The residue was purified by flash columnchromatography (SiO₂, 98:2 to 25:75 cyclohexane/EtOAc) to yield thedesired product.

1.16.5. Method E3

A mixture of Int.2 (1 eq), the aniline (1.1 eq), BINAP (0.3 eq), Cs₂CO₃(4 eq) and Pd(OAc)₂ (0.2 eq) in dry 1,4-dioxane is degassed with aninert gas. The reaction is stirred for a period ranging from 4 h toapproximately 16 h at 100-110° C. The mixture may be partitioned betweenan organic solvent and an aqueous phase. The two layers are separatedand the organic layer is dried and concentrated. Alternatively thereaction mixture can be filtered and concentrated. The residue may bekept as such or purified by chromatography to yield the desired product.

1.16.6. Illustrative Example of Method E3: Synthesis ofN6-(2-Fluoro-4-methanesulfonyl-phenyl)-N4,N4-bis-(4-methoxy-benzyl)-1-methyl-H-benzoimidazole-4,6-diamine(Int.34)

A mixture of Int.2 (1.48 mmol), 2-fluoro-4-methylsulfonyl-aniline (1.58mmol), BINAP (0.47 mmol), Cs₂CO₃ (6.32 mmol) and Pd(OAc)₂ (0.32 mmol) indry 1,4-dioxane (15 mL) was degassed with nitrogen. The reaction wasstirred at 100° C. for approximately 16 h. The mixture was diluted withEtOAc and the organic layer was washed (H₂O), dried (Na₂SO₄) andconcentrated to yield the desired product.

1.16.7. Method E4

A mixture of Int.2 (1 eq), the aniline (1.1 eq), Brettphos (0.1 eq),Cs₂CO₃ (5 eq) and Pd(OAc)₂ (0.05 eq) in dry 1,4-dioxane is degassed withan inert gas. The reaction is stirred for a period ranging from 2 h toapproximately 8 h at 85-95° C. The mixture may be partitioned between anorganic solvent and an aqueous phase. The two layers are separated andthe organic layer is dried and concentrated. Alternatively the reactionmixture can be filtered and concentrated. The residue may be kept assuch or purified by chromatography to yield the desired product.

1.16.8. Illustrative Example of Method E4: Synthesis ofN6-(2,3-dihydro-1,4-benzodioxin-6-yl)-N4,N4-bis[(4-methoxyphenyl)methyl]-1-methyl-benzimidazole-4,6-diamine(Int.78)

A mixture of Int.2 (1.17 mmol), 2,3-dihydro-1,4-benzodioxin-6-amine(1.29 mmol), Brettphos (0.117 mmol), Cs₂CO₃ (6.32 mmol) and Pd(OAc)₂(0.058 mmol) in dry 1,4-dioxane (15 mL) was degassed with nitrogen. Thereaction was stirred at 90° C. for approximately 5 h. The mixture wasdiluted with EtOAc and the organic layer was washed (H₂O), dried(Na₂SO₄) and concentrated to yield the desired product.

1.17. General Method: Methylation of Buchwald Product

Where R_(A) can be optionally substituted aryl or heteroaryl.

1.17.1. Method F

NaH (60% in mineral oil, 1.1 to 3 eq) is added to a solution of theintermediate obtained from the Buchwald coupling (1 eq) in THF or DMF at0° C. The mixture is stirred at 0° C. for 30 min. MeI (1.1 to 3 eq) isadded and the mixture is stirred at room temperature for a periodranging from 1 h to approximately 16 h. The mixture is partitionedbetween an organic solvent and an aqueous phase. The two layers areseparated and the organic layer is dried and concentrated. The residueis purified by silica chromatography or used as such without furtherpurification.

1.17.2. Illustrative Example of Method F: Synthesis ofN6-(2-Fluoro-4-methanesulfonyl-6-methyl-phenyl)-N4,N4-bis-(4-methoxy-benzyl)-1,N6-dimethyl-1H-benzoimidazole-4,6-diamine(Int.37)

NaH (60% in mineral oil, 6.76 mmol) was added to a solution of Int.35(2.25 mmol) in THF (10 mL) at 0° C. The mixture was stirred at 0° C. for30 min. MeI (6.76 mmol) was added and the mixture was stirred at roomtemperature for approximately 16 h. The mixture was diluted with EtOAcand washed with H₂O. The organic layer was dried (Na₂SO₄) andconcentrated. The residue was purified by flash column chromatography(SiO₂, 20:80 to 80:20 EtOAc/petroleum ether) to yield the desiredproduct.

1.17.3. Illustrative Example of Method F: Synthesis ofN4,N4-bis[(4-methoxyphenyl)methyl]-N6,1-dimethyl-N6-(2-methyl-4-methylsulfonyl-phenyl)benzimidazole-4,6-diamine(Int.49A) andN6-(4-ethylsulfonyl-2-methyl-phenyl)-N4,N4-bis[(4-methoxyphenyl)methyl]-N6,1-dimethyl-benzimidazole-4,6-diamine(Int.49B)

NaH (60% in mineral oil, 4.86 mmol) was added to a solution of Int.36(1.62 mmol) in THF (10 mL) at 0° C. The mixture was stirred at 0° C. for30 min. MeI (4.86 mmol) was added and the mixture was stirred at roomtemperature for 1 h. The mixture was diluted with EtOAc and washed withH₂O. The organic layer was dried (Na₂SO₄) and concentrated. Flash columnchromatography (SiO₂, 20:80 to 80:20 EtOAc/petroleum ether) yielded amixture of the desired product (Int.49A, major product) together withthe a side product (Int.49B, minor product).

1.18. Synthesis of4-{7-[Bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-3-ethylbenzonitrile(Int.50)

A mixture of Int.2 (0.4 mmol), Int.22 (0.4 mmol), CuI (0.06 mmol) andCs₂CO₃ (1 mmol) in pyridine (2 mL) was flushed with argon, scaled andheated in a microwave reactor for 3 h at 200° C. The mixture was dilutedwith H₂O and extracted with EtOAc. The organic layer was dried andconcentrated to yield the desired product.

1.19. General Method: Ullmann Coupling Between5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile(Int.3) and aromatic or heteroaromatic halides

Where R_(A) can be optionally substituted aryl or heteroaryl.

1.19.1. Method G

A mixture of Int.3 (1 eq), the aryl halide (1.2 to 1.3 eq), CuI (0.1 to0.2 eq), N,N-dimethyl glycine (0.3 to 0.5 eq) and Cs₂CO₃ (3 eq) in 1:1DMF/1,4-dioxane is flushed with an inert gas and stirred at 110° C. fora period ranging from 4.5 h to 16 h. The mixture is filtered,partitioned between an organic solvent and an aqueous phase. The twolayers are separated and the organic layer is dried and concentrated.The residue is purified by silica chromatography.

1.19.2. Illustrative Example of Method G: Synthesis of5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile(Int.51)

A mixture of Int.3 (0.39 mmol), Int.18 (0.47 mmol), CuI (0.08 mmol),N,N-dimethyl glycine (0.2 mmol) and Cs₂CO₃ (1.17 mmol) in 1:1DMF/1,4-dioxane (4 mL) was flushed with argon and stirred at 110° C. forapproximately 16 h in a sealed tube. The reaction mixture was cooled toroom temperature and filtered through a Celite pad. The filtrate wasconcentrated. Water was added and the mixture was extracted with EtOAc.The organic layer was washed with water, dried and concentrated. Theresidue was purified by flash column chromatography (SiO₂, 100:0 to40:60 cyclohexane/EtOAc) to yield the desired product.

1.20. General Method: SNAr Coupling Between5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile(Int.3) and aromatic or heteroaromatic halides

Where R_(A) can be optionally substituted aryl or heteroaryl.

1.20.1. Method H

A mixture of Int.3 (1 eq), the aryl/heteroaryl halide (1.2 to 1.5 eq)and K₂CO₃ (2 eq) in DMF is stirred at 100° C. for a period ranging from3 h to approximately 16 h. The mixture is diluted with EtOAc, washedseveral times with H₂O, dried and concentrated. The residue is purifiedby flash column chromatography.

1.20.2. Illustrative Example of Method H: Synthesis of5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile(Int.51)

A mixture of Int.3 (58 mmol), Int.7 (70 mmol) and K₂CO₃ (116 mmol) inDMF (160 mL) was stirred at 100° C. After 3 h a further 7.35 mmol ofInt.7 were added to the reaction and the mixture was stirred for anadditional 1 h at 100° C. The resulting mixture was diluted with EtOAc,washed several times with H₂O, dried (Na₂SO₄) and concentrated. Theresidue was purified by flash column chromatography (SiO₂, 15:85 to35:75 EtOAc/petroleum ether) to yield the desired product.

1.21. General Method: Bis-PMB Deprotection

Where R_(A) can be optionally substituted aryl or heteroaryl; and -L-can be: —NH—, —NMe- or —O—.

1.21.1. Method I1

A mixture of protected bis-PMB amine (1 eq) in TFA is stirred at roomtemperature for a period ranging from 30 min to approximately 16 h. Thetemperature is increased to 50 or 60° C. and the mixture is furtherstirred for a period ranging from 0.5 to 3 h. The mixture undergoes workup using a basic aqueous solution and an organic solvent. The two phasesare separated and the organic layer is dried and concentrated.Alternatively, the reaction mixture can be first concentrated and thenthe residue undergoes the above described work up. The residue obtainedfrom the work up can be purified by silica chromatography or by ISOLUTE®SCX-3 (Biotage) ion exchange resin or kept as such.

1.21.2. Illustrative Example of Method I1: Synthesis ofN6-(2-Fluoro-4-methanesulfonyl-6-methyl-phenyl)-1,N6-dimethyl-1H-benzoimidazole-4,6-diamine(Int.53)

A mixture of Int.37 (0.33 mmol) in TFA (5 mL) was stirred at roomtemperature for 1 h and at 50° C. for 0.5 h. the mixture wasconcentrated. The residue was partitioned between sat. NaHCO₃ and DCM.The two phases were separated and the organic layer was dried (Na₂SO₄)and concentrated. The residue was purified by flash columnchromatography (SiO₂, 20:80 to 100:0 EtOAc/petroleum ether) to yield thedesired product.

1.21.3. Illustrative Example of Method I1: Synthesis ofN6,1-dimethyl-N6-(2-methyl-4-methylsulfonyl-phenyl)benzimidazole-4,6-diamine(Int.56A) andN6-(4-ethylsulfonyl-2-methyl-phenyl)-N6,1-dimethyl-benzimidazole-4,6-diamine(Int.56B)

A mixture of Int.49A and 49B (ca 0.61 mmol in total) in TFA (5 mL) wasstirred at room temperature for approximately 16 h and at 50° C. for 3h. the mixture was concentrated. The residue was partitioned betweensat. NaHCO₃ and DCM. The two phases were separated and the organic layerwas dried (Na₂SO₄) and concentrated. Flash column chromatography (SiO₂,20:80 to 100:0 EtOAc/petroleum ether) yielded a mixture of the desiredproduct (Int.56A, major product) together with the a side product(Int.56B, minor product) deriving from the deprotection of Int.49B.

1.21.4. Method I2

A mixture of protected bis-PMB amine (1 eq) in DCM/TFA (3:1 to 1:1ratio) is stirred at room temperature for a period ranging from 20 minto 3 h. The mixture undergoes work up using a basic aqueous solution andan organic solvent. The two phases are separated and the organic layeris dried and concentrated. Alternatively, the reaction mixture can befirst concentrated and then the residue undergoes the above describedwork up. The residue obtained from the work up can be purified by silicachromatography or by ISOLUTE® SCX-3 (Biotage) ion exchange resin or keptas such.

1.21.5. Illustrative Example of Method I2: Synthesis of5-(7-amino-3-methyl-benzimidazol-5-yl)oxy-4-methyl-pyridine-2-carbonitrile(Cpd 28)

TFA (55 mL) was added to a solution of Int.51 (23.1 mmol) in DCM (55 mL)at 0° C. The mixture was stirred at room temperature for 20 min. Toluenewas added and the mixture was concentrated. The residue was partitionedbetween DCM and sat. NaHCO₃. The two phases were separated and theorganic layer was dried (Na₂SO₄) and concentrated. The residue waspurified by flash column chromatography (SiO₂, 100:0 to 95:5EtOAc/MeOH). The product obtained from the column was washed with THF toyield the desired product (Int 79).

1.21.6. Method I3

A mixture of protected bis-PMB amine (1 eq) in TFA is stirred at roomtemperature for a period ranging from 30 min to approximately 16 h. Themixture is diluted in an organic solvent and washed using a basicaqueous solution. The two phases are separated and the organic layer isdried and concentrated. Alternatively, before washing, the reactionmixture can be first concentrated. The organic layer is thenconcentrated, and the residue can be either purified by silicachromatography or by ISOLUTE® SCX-3 (Biotage) ion exchange resin or usedas such. without any further purification

1.21.7. Illustrative Example of Method I3: Synthesis of5-[(7-amino-3-methyl-3H-benzoimidazol-5-yl)-methyl-amino]-4-methyl-pyridine-2-carbonitrile(Int.62)

A solution of Int.41 (2.4 mmol) in TFA (10 mL) was stirred at roomtemperature for 3 h. The mixture was diluted (DCM), washed with sat.NaHCO₃ and then with 5 N NaOH. The two layers were separated and theaqueous layer was further extracted with DCM. The organic layers werecombined, dried (filtration through phase separator) and concentrated.The residue was purified by flash column chromatography (SiO₂, 100:0 to90:10 EtOAc/MeOH) to yield the desired product.

1.22. Synthesis of 4-Ethyl-6-methanesulfonyl-pyridin-3-ylamine (Int.70)

1.22.1. Step i: 4-ethyl-2-methylsulfonyl-5-nitro-pyridine

A mixture of 2-bromo-4-ethyl-5-nitro-pyridine (4.35 mmol) and sodiummethanesulfinate (4.35 mmol) in DMSO (10 mL) was stirred at roomtemperature for 1.5 h. The mixture was poured into ice-water and stirreduntil the ice had melted. The mixture was filtered and the solid (thedesired product) was collected.

1.22.2. Step ii: 4-ethyl-6-methylsulfonyl-pyridin-3-amine

A mixture of 4-ethyl-2-methylsulfonyl-5-nitro-pyridine (4.1 mmol), NH₄Cl(26.65 mmol) and iron powder (33 mmol) in H₂O (10 mL) was stirred at 90°C. for 1 h. The reaction was stopped, filtered and extracted with EtOAc.The organic layer was dried (Na₂SO₄) and concentrated to yield thedesired product.

1.23. Synthesis of 4-ethyl-5-iodo-2-methylsulfonyl-pyridine (Int.74)

A solution of KI (15 mmol) and NaNO₂ (12 mmol) in H₂O (1.8 mL) was addeddropwise to a mixture of Int.70 (6 mmol) and p-TsOH.H₂O (18 mmol) inMeCN (12 mL) keeping the temperature at 10 to 15° C. The mixture wasstirred for 10 min at 10 to 15° C. and then at room temperature for 1 h.The mixture was cooled to 0° C., neutralized (sat. NaHCO₃) and extracted(DCM). The organic layer was dried (Na₂SO₄) and concentrated. Theresidue was purified by flash column chromatography (SiO₂, 70:30petroleum ether/EtOAc) to yield the desired product.

Example 2 Synthesis of the Compounds of the Invention 2.1. GeneralMethod: Acylation of the Amine Intermediate with a Carbonyl ChlorideDerivative to Obtain a Final Compound

Where R_(A) can be optionally substituted aryl or heteroaryl; L can beNH, NMe or O; and R_(B) can be cycloalkyl, OMe.

2.1.1. Method J1

R_(B)COCl (1 eq) is added to a solution of the intermediate amine (1 eq)in DCM/pyridine (from 2:1 to 5:1 ratio) at 0° C. The mixture is stirredfor a period ranging from 40 min to 2 h. The mixture is partitionedbetween an organic solvent and an aqueous solution. The two phases areseparated and the organic layer is dried and concentrated.Alternatively, the reaction mixture can be concentrated withoutundergoing work up. The residue can be purified by silica chromatographyor by preparative HPLC or by precipitation using the appropriate solventmixture.

2.1.2. Illustrative Example of Method J1: Synthesis ofN-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide(Cpd 1)

Cyclopropanecarbonyl chloride (0.39 mmol) was added to a solution ofInt.63 (0.39 mmol) in 1:2 pyridine/DCM (2.55 mL) at 0° C. The mixturewas stirred at room temperature for 40 min. The reaction mixture wasconcentrated and the residue was purified by flash column chromatography(SiO2, 100:0 to 97:3 DCM/MeOH) to afford the desired product.

2.1.3. Method J2

R_(B)C(═O)Cl (1.5 to 3 eq) is added to a solution of the intermediateamine (1 eq) in DCM followed by pyridine (1.5 to 3 eq). The mixture isstirred for a period ranging from 2 h to approximately 16 h. The mixtureis partitioned between an organic solvent and an aqueous solution. Thetwo phases are separated and the organic layer is dried andconcentrated. The residue can be purified by silica chromatography, bypreparative HPLC or by precipitation using the appropriate solventmixture.

2.1.4. Illustrative Example of Method J2: Synthesis of methyl6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate(Cpd 4)

Methyl chloroformate (0.33 mmol) was added to a solution of Int.63 (0.22mmol) in dry DCM (2.2 mL) followed by pyridine (0.33 mmol). The mixturewas stirred at room temperature for 2 h. The mixture was partitionedbetween H₂O and DCM. The two phases were separated and the organic layerwas washed (sat. NaHCO₃), dried (Na₂SO₄) and concentrated. The residuewas purified by flash column chromatography (SiO₂, 100:0 to 95:5DCM/MeOH) to yield the desired product.

2.2. General Method: Acylation the Amine Intermediate with a CarboxylicAcid Derivative to Obtain a Final Compound

Where R_(A) can be optionally substituted aryl or heteroaryl; L can be:NH, NMe or O; and R_(C) can be: cycloalkyl, substituted cycloalkyl.

2.2.1. Method K1

(COCl)₂ (1.4 to 2 eq) is added to a solution of the carboxylic acid (1.5to 2 eq) in DCM at 0° C. A catalytic amount of DMF is added and thereaction is stirred at 0° C. for a period ranging from 30 min to 1 h. Asolution of the intermediate amine (1 eq) and pyridine (2 to 4 eq) inDCM is added to the mixture. The resulting mixture is stirred at roomtemperature for a period ranging from 30 min to 2 h. The mixture ispartitioned between an organic solvent and an aqueous solution. The twophases are separated and the organic layer is dried and concentrated.The residue can be purified by silica chromatography, by preparativeHPLC or by precipitation using the appropriate solvent mixture.

2.2.2. Illustrative Example of Method K1: Synthesis of(1R,2R)-N-[6-[(6-cyano-4-ethyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide(Cpd 6)

(COCl)₂ (0.186 mmol) was added to a solution of((1R,2R)-(−)-cis-2-fluoro-cyclopropanecarboxylic acid (ChemCollect, lotn. 1241399, 0.186 mmol) in DCM (1 mL) followed by DMF (2 drops). Thereaction mixture was left to stir at 0° C. for 1 h. A solution of Int.65(0.093 mmol) and pyridine (0.186 mmol) in DCM (1 mL) was added to themixture and the reaction was stirred for 2 h at room temperature. Sat.NaHCO₃ was added and the two phases were separated. The organic layerwas concentrated. The residue was purified by flash columnchromatography (SiO₂, 100:0 to 95:5 DCM/MeOH) to yield the desiredproduct.

2.2.3. Illustrative Example of Method K1: Synthesis of(1R,2R)-N-[6-(N,2-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide(Cpd 26) and(1R,2R)-N-[6-(4-ethylsulfonyl-N,2-dimethyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide(Cpd 27)

(COCl)₂ (0.82 mmol) was added to a solution of((1R,2R)-(−)-cis-2-fluoro-cyclopropanecarboxylic acid (ABCR, lot n.1242863, 0.92 mmol) in DCM (1 mL) followed by DMF (1 drop). The reactionmixture was left to stir at 0° C. for 45 min. A solution of a mixture ofInt.56A and Int.56B (approximately 0.46 mmol in total) and pyridine(1.85 mmol) in DCM (1 mL) was added to the mixture and the reaction wasstirred for 2 h at room temperature. The mixture was diluted with DCM,washed (H₂O) and the two phases were separated. The organic layer wasdried (Na₂SO₄) and concentrated. Flash column chromatography (SiO₂,20:80 EtOAc/petroleum ether to 90:10 EtOAc/MeOH) yielded a mixture ofCompound 26 and Compound 27. The two components were separated by prep.HPLC.

2.2.4. Method K2

(COCl)₂ (1.35 to 2.5 eq) is added to a solution of the carboxylic acid(1.4 to 3 eq) in DCM at 0° C. A catalytic amount of DMF is added and thereaction is stirred at 0° C. for a period ranging from 30 min to 1 h. Asolution of the intermediate amine (1 eq) and pyridine (1.7 to 4 eq) inNMP or NMP/DCM is prepared separately making sure that the intermediateamine is fully dissolved. To help dissolution, heating can be applied(temperatures up to 70° C.). The latter solution is added dropwise tothe solution containing the newly formed acyl chloride at 0° C. Theresulting mixture is left to stir at room temperature for a periodranging from 1 to 2 h. The mixture is partitioned between an organicsolvent and an aqueous solution. The two phases are separated and theorganic layer is dried and concentrated. The residue can be purified bysilica chromatography, by preparative HPLC or by precipitation using theappropriate solvent mixture.

2.2.5. Illustrative Example of Method K2: Synthesis of(1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide(Cpd 20)

(COCl)₂ (16.6 mmol) was added to a solution of((1R,2R)-(−)-cis-2-fluoro-cyclopropanecarboxylic acid (ABCR, lot n.1242863, 17.5 mmol) in DCM (70 mL) followed by DMF (200 μL). Thereaction mixture was left to stir at 0° C. for 45 min. A solution ofCompound 28 (12.5 mmol) and pyridine (21.3 mmol) in NMP (15 mL) washeated at 65° C., to help dissolution, and let cool down to roomtemperature. The solution containing Compound 28 and pyridine was addedto the mixture containing the activated carboxylic derivative at 0° C.and the reaction was stirred for 45 min at room temperature. The mixturewas partitioned between EtOAc and sat. NaHCO₃. The two phases wereseparated and the organic layer was washed further (sat. NaHCO₃, NH₄Cl,H₂O), dried (Na₂SO₄) and concentrated. The crude was purified byprecipitation from DCM/iPrOH and the resulting solid was washed withEt₂O and dried under vacuum to afford the desired product.

2.3. General Method: Hydrolysis of a Nitrile Group to Obtain a FinalCompound

Where L is NH, NMc or O; R_(C) is cycloalkyl, substituted cycloalkyl; Zis N or CH; and R¹⁰ is Me or Et.

2.3.1. Method L

A solution of the nitrile starting material (1 eq) is dissolved in a 4:1EtOH/DMSO mixture. The resulting organic solution is mixed with 30%H₂O₂/H₂O and 1 N NaOH with the following proportions 10:2:1 organicsolution/H₂O₂/1N NaOH. The mixture is stirred at 50° C. for 1 h. Themixture is partitioned between an organic solvent and an aqueoussolution. The two phases are separated and the organic layer is driedand concentrated. The residue can be purified by silica chromatography,by preparative HPLC or by precipitation using the appropriate solventmixture.

2.3.2. Illustrative Example of Method L: Synthesis of5-((4-(cyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino)-4-ethylpicolinamide(Cpd 24)

A solution of compound 1 (0.19 mmol) was dissolved in a 4:1 EtOH/DMSOmixture (2.3 mL). The resulting organic solution was mixed with 30%H₂O₂/H₂O (0.4 mL) and 1 N NaOH (0.23 mL). The mixture was stirred at 50°C. for 1 h. The reaction mixture was partitioned between dichloromethaneand water. The layers were separated and the organic layer was filteredthrough a phase separator and concentrated. The residue was purified bypreparative HPLC to yield the desired product.

2.3.3. Method M: General Method of Synthesis of Urea

Carbonylditriazol (1.5 eq.) is added to a mixture of Cpd 28 (1.0 eq.)and pyridine (5 eq;) in DCM. The mixture is then stirred at 50° C. for 1h. Without any further treatment the required amine R^(a)NH₂ is added tothe solution at 50° C. and allowed to stir for another 1 h. Aftercompletion of the reaction by UPLC, the reaction mixture is cooled toroom temperature and diluted with DCM, and the mixture is thenpartitioned in water. The two phases are separated and the organic layeris washed with 0.25 M HCl solution, sodium bicarbonate solution, driedand concentrated. The residue can be purified by silica chromatography,by preparative HPLC or by precipitation using the appropriate solventmixture

2.3.4. Illustrative Example of Method M: Synthesis of1-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl-urea(Cpd 33)

Carbonylditriazol (1.07 mmol) was added to a mixture of Cpd 28 (0.71mmol) and pyridine (3.55 mmol) in DCM (3 mL) and the resulting mixturewas stirred at 50° C. A precipitate formed after 2 min and the mixturewas further stirred for 1 h. Methyl amine (2M solution in THF) (2.84mmol) was then added to the solution at 50° C. and allowed to stir foranother 1 h. After completion of the reaction by UPLC, the reactionmixture was cooled to room temperature and was diluted with DCM andpartitioned in water. The two phases were separated and the organiclayer was washed with 0.25 M HCl solution, sodium bicarbonate solution,dried and concentrated. The residue the used without any furtherpurification.

2.4. Synthesis of5-[7-[[(1R,2R)-2-fluorocyclopropanecarbonyl]amino]-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carboxamide(Cpd 32)

A mixture of Compound 20 (0.27 mmol) and DMSO (0.5 mL) in H₂O (pH 13, 10mL) was stirred at 50° C. for 72 h. The mixture was filtered. The solidwas collected and purified by preparative HPLC to yield the desiredproduct.

2.5. Synthesis of4-methyl-5-[3-methyl-7-(methylamino)benzimidazol-5-yl]oxy-pyridine-2-carbonitrile(Cpd 34) and5-[7-(dimethylamino)-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carbonitrile(Cpd 35)

NaH was added to a mixture of Cpd 28 (0.36 mmol) in THF (10 mL) at 0° C.After 30 min, MeI (0.36 mmol) was added and the reaction mixture wasallowed to warm to room temperature and stirred until completion. Aftercompletion monitored by UPLC, the reaction was diluted in ethyl acetateand washed with water. The organic layer was dried over Na₂SO₄, filteredand evaporated. Cpd 34 and Cpd 35 were isolated by preparative HPLC.

Illustrative compounds of the invention listed in Table III below andcomparative examples have been prepared according to the syntheticmethods described herein using the intermediates listed in Table II. TheNMR spectral data of the compounds of the invention and some of thecomparative examples is given in Table IV.

TABLE II Illustrative intermediates towards the compounds of theinvention Int# Structure SM Mtd MW MS Ms′d  1

4-Methoxy benzylamine Dsc’d 257.3 258.1 (M + 1)  2

Int. 1 + 5- bromo-3- fluoro-N- methyl-2- nitro-aniline Dsc’d- 466.4465.9/ 467.9 (M + 1)  3

Int. 2 Dsc’d- 403.5 404.4 (M + 1)  4

4-amino-3- fluoro- benzonitrile A1 262.0 261.5 (M − 1)  5

Int. 11 A2 263.0 263.9 (M + 1)  6

Int. 23 A1 272.4 272.8 (M + 1)  7

2-chloro-5- fluoro-4- methyl- pyridine B 136.1 137.1 (M + 1)  8

Int. 20 B 133.2 134.1 (M + 1)  9

Int. 13 B 151.1 152.0 (M + 1) 10

6-bromo-4- ethyl-pyridin- 3-amine B 147.2 148.0 (M + 1) 11

Int. 15 B 137.1 138.0 (M + 1) 12

Int. 5 C 151.1 152.0 (M + 1) 13

Int. 6 C 160.6 161.0 (M + 1) 14

Int. 16 C 203.2 204.0 (M + 1) 15

2- fluoropyridin- 3-amine Dsc’d 191.0 190.9/ 192.9 (M + 1) 16

2-fluoro-4- methylsulfonyl- aniline Dsc’d 268.1 267.0/ 268.9 (M + 1) 17

Int. 4 Dsc’d 164.2 165.5 (M + 1) 18

Int. 8 D 244.0 245.0 (M + 1) 19

Int. 10 D 258.1 259.1 (M + 1) 20

2-bromo-4- methyl-5- nitro-pyridine Dsc'd 187.0 187.0/ 189.0 (M + 1) 21

4-amino-3- fluoro- benzonitrile Dsc’d 170.6 Mass could not be detectedby various methods 22

4-amino-3- ethyl- benzonitrile Dsc’d 147.2 146.1 (M − 1) 23

2-chloro-3- fluoro- pyridine-4- carboxylic acid Dsc’d 146.6 147.0(M + 1) 24

Int. 2 + Int. 10 E1 532.6 533.2 (M + 1) 25

Int. 2 + Int. 17 E1 549.6 550.4 (M + 1) 26

Int. 2 + 6- fluoro-4- methyl- pyridin-3- amine E1 511.6 512.5 (M + 1) 27

Int. 2 + Int. 8 E2 518.6 519.4 (M + 1) 28

Int. 2 + Int. 21 E2 556.0 556.3 (M + 1) 29

Int. 2 + 2,6- difluoropyridin- 3-amine E2 515.6 516.4 (M + 1) 30

Int. 2 + Int. 11 E2 522.6 523.3 (M + 1) 31

Int. 2 + Int. 12 E2 536.6 537.0 (M + 1) 32

Int. 2 + Int. 9 E2 536.6 537.0 (M + 1) 33

Int. 2 + 4- amino-3- fluoro- benzonitrile E2 521.6 522.4 (M + 1) 34

Int. 2 + 2- fluoro-4- methylsulfonyl- aniline E3 574.7 575.0 (M + 1) 35

Int. 2 + Int. 14 E3 588.7 589.2 (M + 1) 36

Int. 2 + 2- fluoro-4- methylsulfonyl- aniline E3 570.7 571.4 (M + 1) 37

Int. 35 F 602.7 603.3 (M + 1) 38

Int. 24 F 546.7 547.2 (M + 1) 39

Int. 25 F 563.7 564.3 (M + 1) 40

Int. 26 F 525.6 526.4 (M + 1) 41

Int. 27 F 532.6 533.5 (M + 1) 42

Int. 28 F 570.1 570.4 (M + 1) 43

Int. 29 F 529.6 530.5 (M + 1) 44

Int. 30 F 536.6 536.5 (M + 1) 45

Int. 31 F 550.6 551.0 (M + 1) 46

Int. 32 F 550.6 551.0 (M + 1) 47

Int. 33 F 535.6 522.4 (M + 1) 48

Int. 34 F 588.7 589.5 (M + 1) 49A

Int. 36 F 584.7 585.1 (M + 1) 49B

Int. 36 F (by product) 598.8 599.1 (M + 1) 50

Int. 2 + Int. 22 Dsc’d 532.6 533.4 (M + 1) 51

Int. 3 + Int. 18 (method G), Int. 3 + Int. 7 (method H) G or H 519.6520.4 (M + 1) 52

Int. 3 + Int. 19 G 533.6 534.4 (M + 1) 53

Int. 37 I1 362.4 363.1 (M + 1) 54

Int. 25 I1 309.3 310.3 (M + 1) 55

Int. 43 I1 289.3 290.0 (M + 1) 56A

Int. 49A I1 344.4 344.9 (M + 1) 56B

Int. 49B I1 358.5 359.0 (M + 1) 57

Int. 45 I1 310.3 311.0 (M + 1) 58

Int. 47 I1 295.3 296.0 (M + 1) 59

Int. 48 I1 348.4 349.0 (M + 1) 61

Int. 39 I2 323.4 324.3 (M + 1) 62

Int. 41 I3 292.3 293.3 (M + 1) 63

Int. 38 I3 306.4 307.1 (M + 1) 64

Int. 50 I3 292.3 293.3 (M + 1) 65

Int. 52 I3 293.3 294.2 (M + 1) 66

Int. 42 I3 329.8 330.2 (M + 1) 67

Int. 40 I3 285.3 286.2 (M + 1) 68

Int. 44 I3 296.3 297.0 (M + 1) 69

Int. 32 I3 310.3 311.0 (M + 1) 70

2-bromo-4- ethyl-5-nitro- pyridine Dsc’d 200.3 201.1 (M + 1) 71

Int. 70 + Int. 2 E2 585.7 586.0 (M + 1) 72

Int. 71 F 599.8 600.1 (M + 1) 73

Int. 72 I3 359.5 360.0 (M + 1) 74

Int. 70 Dsc’d 311.1 311.8 (M + 1) 75

Int. 3 + Int. 74 G 586.7 587.3 (M + 1) 76

Int. 75 I2 346.4 347.0 (M + 1) 77

Int. 2 + 2-(4- aminophenyl) acetonitrile E4 517.6 518.0 (M + 1) 78

Int. 2 + 2,3- dihydro-1,4- benzodioxin- 6-amine E4 536.6 537.33 (M + 1)79

Int. 77 I2 277.3 278.0 (M + 1) 80

Int. 78 I2 296.3 297.0 (M + 1)

TABLE III Illustrative compounds of the invention MS Cpd # StructureName SM Mtd MW Ms′d  1

N-(6-((6-cyano-4- ethylpyridin-3- yl)(methyl)amino)-1- methyl-1H-benzo[d]imidazol-4- yl)cyclopropanecarbox- amide Int 63 J1 374.4 375.3 2

N-(6-(4-cyano-2-ethyl- 6-fluorophenylamino)- 1-methyl-1H-benzo[d]imidazol-4- yl)cyclopropanecarbox- amide Int 54 J1 377.4 378.0 3

N-(6-((4-cyano-2- ethyl-6- fluorophenyl)(methyl) amino)-1-methyl-1H-benzo[d]imidazol-4- yl)cyclopropanecarbox- amide Int 61 J1 391.4 392.4 4

methyl 6-((6-cyano-4- ethylpyridin-3- yl)(methyl)amino)-1- methyl-1H-benzo[d]imidazol-4- ylcarbamate Int 63 J2 364.4 365.1  5

methyl 6-((4-cyano-2- ethyl-6- fluorophenyl)(methyl) amino)-1-methyl-1H-benzo[d]imidaxol-4- ylcarbamate Int 61 J2 381.4 382.1  6

(1R,2R)-N-[6-[(6- cyano-4-ethyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]-2-fluoro- cyclopropanecarbox- amide Int 65 K1 379.4 380.4  7

N-(6-((4-cyano-2- ethyl-6- fluorophenyl)(methyl) amino)-1-methyl-1H-benzo[d]imidazol-4- yl)-2- fluorocyclopropane- carboxamide(1S,2S)/(1R,2R) racemic mixture Int 61 K1 409.4 410.2  8

(1R,2R)-N-(6-((6- cyano-4-ethylpyridin- 3-yl)(methyl)amino)-1-methyl-1H- benzo[d]imidazol-4- yl)-2- fluorocyclopropane- carboxamideInt 63 K1 392.4 393.1  9

(1R,2R)-N-(6-((6- cyano-4- methylpyridin-3- yl)(methyl)amino)-1-methyl-1H- benzo[d]imidazol-4- yl)-2- fluorocyclopropane- carboxamideInt 62 K1 378.4 378.9 10

(1R,2R)-N-[6-(4- cyano-2-ethyl- phenoxy)-1-methyl- benzimidazol-4-yl]-2-fluoro- cyclopropanecarbox- amide Int 64 K1 378.4 379.4 11

(1R,2R)-N-[6-(2- chloro-4-cyano-6- fluoro-N-methyl- anilino)-1-methyl-benzimidazol-4-yl]- 2-fluoro- cyclopropanecarbox- amide Int 66 K1 415.8416.0 12

(1R,2R)-2-fluoro-N- [6-[(6-fluoro-4- methyl-3-pyridyl)- methyl-amino]-1-methyl-benzimidazol- 4-yl]cyclopropanecarbox- amide Int 67 K1 371.4372.5 13

(1R,2R)-N-[6-[(2,6- difluoro-3-pyridyl)- methyl-amino]-1-methyl-benzimidazol- 4-yl]-2-fluoro- cyclopropanecarbox- amide Int 55 K1375.4 376.1 14

(1R,2R)-N-[6-[(6- cyano-2-fluoro-3- pyridyl)-methyl- amino]-1-methyl-benzimidazol-4-yl]-2- fluoro-cyclopropane- carboxamide Int 68 K1 382.4383.0 15

(1R,2R)-N-[6-(4- cyano-2-fluoro-N- methyl-anilino)-1-methyl-benzimidazol- 4-yl]-2-fluoro- cyclopropanecarbox- amide Int 58 K1381.4 382.1 16

(1R,2R)-2-fluoro-N- [6-(2-fluoro-N,6- dimethyl-4- methylsulfonyl-anilino)-1-methyl- benzimidazol-4-yl] cyclopropanecarbox- amide Int 53K1 448.5 448.9 17

(1R,2R)-2-fluoro-N- [6-(2-fluoro-N-methyl- 4-methylsulfonyl-anilino)-1-methyl- benzimidazol-4-yl] cyclopropanecarbox- amide Int 59K1 435.5 435.1 18

N-[6-[(4-ethyl-6- methylsulfonyl-3- pyridyl)-methyl- amino]-1-methyl-benzimidazol-4-yl] cyclopropanecarbox- amide Int 73 K1 427.5 428.2 19

N-[6-(2-fluoro-N,6- dimethyl-4- methylsulfonyl- anilino)-1-methyl-benzimidazol-4-yl] cyclopropanecarbox- amide Int 53 K1 430.5 431.0 20

(1R,2R)-N-[6-[(6- cyano-4-methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]-2-fluoro- cyclopropanecarbox- amide Cpd 28 K2 365.4 366.0 21

(1R,2R)-N-[6-[(6- cyano-2-fluoro-4- methyl-3-pyridyl)- methyl-amino]-1-methyl-benzimidazol- 4-yl]-2-fluoro- cyclopropanecarbox- amide Int 57 K2396.4 397.1 22

(1R,2R)-N-[6-[(2- cyano-3-fluoro-5- methyl-4-pyridyl)- methyl-amino]-1-methyl-benzimidazol- 4-yl]-2-fluoro- cyclopropanecarbox- amide Int 69 K2396.4 397.1 23

N-[6-[(6-cyano-4- methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]cyclopropanecarbox- amide Cpd 28 K2 347.4 348.1 24

5-((4- (cyclopropanecarbox- amido)-1-methyl-1H- benzo[d]imidazol-6-yl)(methyl)amino)-4- ethylpicolinamide Cpd 1 L 392.5 393.3 25

4-ethyl-5-((4- ((1R,2R)-2- fluorocyclopropane- carboxamido)-1-methyl-1H-benzo[d]imidazol- 6-yl)(methyl)amino) picolinamide Cpd 8 L 410.5411.3 26

(1R,2R)-N-[6-(N,2- dimethyl-4- methylsulfonyl- anilino)-1-methyl-benzimidazol-4-yl]- 2-fluoro- cyclopropanecarbox- amide Int 56A — 430.5431.0 27

(1R,2R)-N-[6-(4- ethylsulfonyl-N,2- dimethyl-anilino)-1-methyl-benzimidazol- 4-yl]-2-fluoro- cyclopropanecarbox- amide Int 56BK1 444.5 445.0 28

5-(7-amino-3-methyl- benzimidazol-5- yl)oxy-4-methyl-pyridine-2-carbonitrile Int 51 I2 279.3 280.1 29

N-[6-[(4-ethyl-6- methylsulfonyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]cyclopropanecarbox- amide Int 76 K1 414.5 415.1 30

N-[6-[4- (cyanomethyl)anilino]- 1-methyl- benzimidazol-4-yl]cyclopropanecarbox- amide Int 79 K1 345.4 346.0 31

N-[6-(2,3-dihydro-1,4- benzodioxin-6- ylamino)-1-methyl- benzimidazol-4-yl]cyclopropanecarbox- amide Int 80 K1 364.4 365.0 32

5-[7-[[(1R,2R)-2- fluorocyclopropane- carbonyl]amino]-3-methyl-benzimidazol- 5-yl]oxy-4-methyl- pyridine-2- carboxamide Cpd 20Dsc’d 383.4 384.1 33

1-[6-[(6-cyano-4- methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]-3-isopropyl-urea Int 83 M 336.4 337.0 34

4-methyl-5-[3-methyl- 7-(methylamino) benzimidazol-5-yl]oxy-pyridine-2-carbonitrile Int 83 Dsc’d 293.3 294.0 35

5-[7-(dimethylamino)- 3-methyl- benzimidazol-5- yl]oxy-4-methyl-pyridine-2-carbonitrile Int 83 Dsc’d 307.4 308.0 36

N-[6-[(6-cyano-4- methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]-3-hydroxy- azetidine-1- carboxamide Int 83 M 378.1 379.0 37

N-[6-[(6-cyano-4- methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]morpholine-4- carboxamide Int 83 M 392.2 393.1 38

1-[6-[(6-cyano-4- methyl-3- pyridyl)oxy]-1- methyl-benzimidazol-4-yl]-3-isopropyl-urea Int 83 M 364.2 365.1

TABLE IV NMR data of illustrative compounds of the invention Cpd# NMR 1¹H NMR (600 MHz, DMSO-d₆): d = 0.69-0.77 (m, 4H), 1.04 (t, 3H),2.15-2.24 (m, 1H), 2.34 (q, 2H), 3.34 (s, 3H), 3.74 (s, 3H), 6.72 (d,1H), 7.49 (s, 1H), 7.94 (s, 1H), 8.04 (s, 1H), 8.48 (s, 1H), 10.05 (s,1H). 2 ¹H NMR (400 MHz, DMSO-d₆): δ = 0.70-0.80 (m, 4H), 1.09 (t, 3H),2.19-2.27 (m, 1H), 2.64 (q, 2H), 3.68 (s, 3H), 6.42 (t, 1H), 7.55 (d,1H), 7.64 (dd, 1H), 7.67 (br.s, 1H), 7.92 (s, 1H), 7.98 (s, 1H), 10.01(s, 1H). 3 ¹H NMR (300 MHz, DMSO-d₆): d = 9.93 (s, 1H), 7.96 (s, 1H),7.81 (dd, 1H), 7.74 (s, 1 H), 7.29 (br s, 1H), 6.47 (d, finely split,1H), 3.73 (s, 3H), 3.21 (s, 3H), 2.54-2.48 (m, partially obscured bysolvent peak, 2H), 2.17 (m, 1H), 1.09 (t, 3H), 0.71-0.69 (m, 4H). 4 ¹HNMR (300 MHz, DMSO-d₆): δ = 1.05 (t, 3H), 2.36 (q, 2H), 3.36 (s, 3H),3.61 (s, 3H), 3.73 (s, 3H), 6.72 (d, 1H), 7.06 (d, 1H), 7.97 (s, 1H),8.00 (dd, 1H), 8.50 (s, 1H), 8.91 (bs, 1H). 5 ¹H NMR (300 MHz, DMSO-d₆):δ = 1.10 (t, 3H), 2.52 (q, 2H), 3.23 (s, 3H), 3.59 (s, 3H), 3.73 (s,3H), 6.51 (d, 1H), 6.86 (d, 1H), 7.76 (bs, 1H), 7.82 (dd, 1H), 7.92 (s,1H), 8.75 (s, 1H). 6 ¹H NMR (400 MHz, DMSO-d₆): d = 1.14 (ddt, H), 1.24(t, 3H), 1.53-1.67 (m, 1H), 2.51-2.55 (m, 1H), 2.75 (q, 2H), 3.79 (s,3H), 4.77-5.03 (m, 1H), 7.13 (d, 1H), 7.85 (d, 1H), 8.07 (s, 1H), 8.11(s, 1H), 8.22 (s, 1H), 10.43 (s, 1H). 7 ¹H NMR (300 MHz, DMSO-d₆): δ =0.99-1.15 (m, 1H), 1.09 (t, 3H), 1.54 (m, 1H), 2.39 (m, 1H), 2.52 (q,2H), 3.22 (s, 3H), 3.73 (s, 3H), 4.85 (m, 1H), 6.49 (d, 1H), 7.32 (s,1H), 7.75 (s, 1H), 7.82 (dd, 1H), 7.96 (s, 1H), 9.96 (s, 1H). 8 ¹H NMR(300 MHz, DMSO-d₆): δ = 0.99-1.18 (m, 1H), 1.05 (t, 3H), 1.56 (m, 1H),2.29-2.45 (m, 1H), 2.35 (q, 2H), 3.35 (s, 3H), 3.74 (s, 3H), 4.88 (m,1H), 6.72 (d, 1H), 7.51 (bs, 1H), 7.95 (s, 1H), 8.05 (s, 1H), 8.49 (s,1H), 10.09 (bs, 1H). 9 ¹H NMR (300 MHz, DMSO-d₆): d = 10.13 (s, 1H),8.49 (s, 1H), 8.07 (s, 1H), 7.87 (s, 1H), 7.55 (br s, 1H), 6.79 (d,finely split, 1H), 5.0-4.7 (m, 1H), 3.75 (s, 3H), 3.38 (s, 3H), 2.5-2.3(m, 1H), 1.97 (s, 3H), 1.6-1.5 (m, 1H), 1.2-1.0 (m, 1H). 10 ¹H NMR (500MHz, DMSO-d₆): d = 1.07-1.20 (m, 1H), 1.24 (t, 3H), 1.53-1.67 (m, 1H),2.43-2.48 (m, 1H), 2.75 (q, 2H), 3.80 (s, 3H), 4.78-5.01 (m, 1H), 6.78(d, 1H), 7.11 (d, 1H), 7.59 (dd, 1H), 7.78 (d, 1H), 7.80 (d, 1H), 8.22(s, 1H), 10.40 (s, 1H). 11 ¹H NMR (400 MHz, DMSO-d₆): d = 1.08 (ddt,1H), 1.41-1.67 (m, 1H), 2.41 (dt, 1H), 3.27 (s, 3H), 3.75 (s, 3H),4.65-5.05 (m, 1H), 6.61 (d, 1H), 7.42 (s, 1H), 8.00 (s, 1H), 8.04 (dd,1H), 8.12 (t, 1H), 10.04 (s, 1H). 12 ¹H NMR (500 MHz, DMSO-d₆): δ = 1.08(ddt, 1H), 1.50-1.60 (m, 1H), 2.12 (s, 3H), 2.39 (dt, 1H), 3.25 (s, 3H),3.73 (s, 3H), 4.76-4.96 (m, 1H), 6.49 (d, 1H), 7.19 (s, 1H), 7.35 (s,1H), 7.97 (s, 1H), 8.00 (s, 1H), 10.02 (s, 1H). 13 ¹H NMR (300 MHz,DMSO-d₆): 10.12 (s, 1H), 8.06-8.00 (m, 2H), 7.96 (s, 1H), 7.22-7.19 (m,1H), 6.76 (s, 1H), 4.98-4.77 (d, 1H), 3.76 (s, 3H), 3.25 (s, 3H),2.45-2.42 (m, 1H), 1.59-1.52 (m, 1H), 1.27-1.10 (m, 1H). 14 ¹H NMR (400MHz, DMSO-d₆) 10.30 (1 H, s), 8.20 (1 H, s), 7.92 (1 H, dd), 7.85 (1 H,d), 7.63 (1 H, dd), 7.17 (1 H, d), 4.92 (1H, dF), 3.79 (3 H, s), 3.40 (3H, s), 2.48-2.44 (1H, m), 1.64-1.53 (1H, m), 1.19-1.15 (1H, m). 15 ¹HNMR (300 MHz, DMSO-d₆): 10.22 (1H, s), 8.16 (1H, s), 7.80 (1H, s),7.70-7.67 (1H, m), 7.59-7.58 (1H, d), 7.25-7.21 (1H, m), 7.07 (1H, s),4.99-4.81 (1H, d), 3.79 (3H, s, CH3), 3.38 (3H, s, CH3), 2.46-2.42 (1H,m, CH), 1.61-1.55 (1H, m), 1.19-1.09 (1H, m) 16 ¹H NMR (400 MHz,DMSO-d₆): 10.30 (1 H, s), 7.95 (1 H, s), 7.78 (1 H, d), 7.74 (1 H, dd),7.36 (1 H, d), 6.49 (1 H, d), 4.87 (1H, dF), 3.78 (3 H, s), 3.28 (3 H,s), 3.21 (3 H, s), 2.43-2.38 (1H, m), 2.22 (3 H, s), 1.62-1.51 (1H, m),1.15-1.08 (1H, m). 17 ¹H NMR (400 MHz, DMSO-d₆): 10.84 (1 H, s), 9.21 (1H, s), 7.79 (2 H, dt), 7.55 (1 H, t), 7.42 (1 H, s), 7.26 (1 H, d), 4.95(1H, dF), 3.96 (3 H, s), 3.43 (3 H, s), 3.28 (3 H, s), 2.43-2.38 (1 H,m), 1.62-1.51 (1 H, m), 1.15-1.08 (1 H, m). 18 ¹H NMR (400 MHz,DMSO-d₆): 10.08 (1 H, s), 8.53 (1 H, s), 8.05 (1 H, s), 7.93 (1 H, s),7.48 (1 H, br s), 6.69 (1 H, d), 3.75 (3 H, s), 3.29 (3 H, s), 3.21 (3H, s), 2.46 (2 H, q), 2.25-2.17 (1 H, m), 1.08 (3 H, t), 0.76-0.69 (4 H,m). 19 ¹H NMR (400 MHz, CDCl₃): 9.55 (1 H, br s), 8.05-8.00 (2 H, m),7.70 (1 H, d), 7.61 (1 H, dd), 6.01 (1 H, d), 3.78 (3 H, s), 3.33 (3 H,s), 3.13 (3 H, s), 2.25 (3 H, s), 1.97-1.92 (1 H, m), 0.91-0.85 (4 H,m). 20 ¹H NMR (500 MHz, DMSO-d₆): d = 1.15 (ddt, 1H), 1.54-1.66 (m, 1H),2.36 (s, 3H), 2.52-2.54 (m, 1H), 3.80 (s, 3H), 4.77-5.03 (m, 1H), 7.12(d, 1H), 7.86 (d, 1H), 8.08 (s, 1H), 8.12 (s, 1H), 8.23 (s, 1H), 10.44(s, 1H). 21 ¹H NMR (400 MHz, DMSO-d₆): 10.12 (1H, s), 8.11 (1H, s), 8.02(1H, s), 7.41 (1H, s), 6.61 (1H, d), 4.88 (1H, m), 3.75 (3H, s), 3.26(3H, s), 2.42 (1H, m), 2.22 (3H, s), 1.57 (1H, m), 1.09 (1H, m). 22 ¹HNMR (400 MHz, DMSO-d₆): 10.18 (1H, s), 8.50 (1H, s), 8.06 (1H, s), 7.51(1H, s), 6.73 (1H, d), 4.87 (1H, m), 3.75 (3H, s), 3.32 (3H, s), 2.45(1H, m), 2.11 (3H, s), 1.60 (1H, m), 1.11 (1H, m). 23 ¹H NMR (400 MHz,DMSO-d₆): 10.39 (1 H, s), 8.23 (1 H, s), 8.11 (1 H, s), 8.07 (1 H, s),7.84 (1 H, d), 7.11 (1 H, d), 3.79 (3 H, s), 2.35 (3 H, s), 2.33-2.27 (1H, m), 0.81-0.76 (4 H, m). 24 ¹H NMR (600 MHz, DMSO-d₆): d = 0.56-0.78(m, 4H), 1.09 (t, 3H), 2.18 (br. s., 1H), 2.46 (br. s., 2H), 3.30 (br.s., 3H), 3.73 (br. s., 3H), 6.56 (br. s., 1H), 7.38 (br. s., 1H), 7.59(br. s., 1H), 7.98 (br. s., 2H), 8.05 (br. s., 1H), 8.33 (br. s., 1H),9.96 (br. s., 1H). 25 ¹H NMR (600 MHz, DMSO-d₆): d = 1.05-1.08 (m, 1H),1.09 (t, 3H), 1.50-1.59 (m, 1H), 2.39 (dt, 1H), 2.45-2.49 (m, 2H), 3.31(s, 3H), 3.73 (s, 3H), 4.70-4.97 (m, 1H), 6.57 (d, 1H), 7.42 (s, 1H),7.59 (d, 1H), 7.99 (s, 2H), 8.06 (d, 1H), 8.34 (s, 1H), 10.00 (s, 1H).26 ¹H NMR (400 MHz, DMSO-d₆): 10.10 (1 H, s), 8.04 (1 H, s), 7.78 (1 H,s), 7.76 (1 H, dd), 7.51 (1 H, d), 7.35 (1 H, d), 6.68 (1 H, d), 4.90(1H, d), 3.75 (3 H, s), 3.29 (3 H, s), 3.21 (3 H, s), 2.40 (1H, t), 2.08(3 H, s), 1.64-1.53 (1H, m), 1.15-1.08 (1H, m). 27 ¹H NMR (400 MHz,DMSO-d₆): 10.10 (1 H, s), 8.03 (1 H, s), 7.73-7.71 (2 H, m), 7.48 (1 H,d), 7.38 (1 H, d), 6.68 (1 H, dd), 4.90 (1H, dF), 3.74 (3 H, s), 3.29 (3H, s), 3.23 (2 H, q), 2.40 (1H, t), 2.07 (3 H, s), 1.64-1.53 (1H, m),1.15-1.08 (1H, m) 1.11 (3 H, t). 28 ¹H NMR (400 MHz, DMSO-d₆): 8.09 (1H, s), 8.05 (1 H, s), 7.95 (1 H, s), 6.53 (1 H, d), 6.11 (1 H, d), 5.60(2 H, br s) 3.68 (3 H, s), 2.34 (3 H, s). 29 ¹H NMR (400 MHz, DMSO-d₆):10.42 (1 H, s), 8.23 (1 H, s), 8.13 (1 H, s), 8.03 (1 H, s), 7.86 (1 H,d), 7.13 (1 H, d), 3.80 (3 H, s), 3.24 (3 H, s), 2.83 (2 H, q),2.33-2.27 (1 H, m), 1.26 (3 H, t), 0.81-0.76 (4 H, m). 30 ¹H NMR (400MHz, CDCl₃) 8.81 (1 H, s), 8.02 (1 H, d), 7.71 (1 H, s), 7.17 (2 H, d),7.04 (2 H, d), 6.83 (1 H, d), 6.05 (1 H, s), 3.73 (3 H, s), 3.65 (2 H,s), 1.73-1.66 (1 H, m), 1.10-1.06 (2 H, m), 0.87-0.82 (2 H, m). 31 ¹HNMR (400 MHz, CDCl₃) 8.77 (1 H, s), 7.89 (1 H, s), 7.66 (1 H, s), 6.78(1 H, d), 6.68 (2 H, t), 6.63 (1 H, dd), 5.73 (1 H, br s), 4.25-4.21 (4H, m), 3.69 (3 H, s), 1.73-1.66 (1 H, m), 1.11-1.06 (2 H, m), 0.87-0.82(2 H, m). 32 ¹H NMR (400 MHz, DMSO-d₆): 10.41 (1 H, br s), 8.20 (1 H,s), 8.07 (1 H, s), 8.03 (1 H, s), 7.98 (1 H, br s), 7.84 (1 H, d), 7.55(1 H, br s), 7.01 (1 H, d), 5.02-4.80 (1 H, m), 3.78 (3 H, s), 2.34 (3H, s), 1.66-1.55 (1 H, m), 1.21-1.02 (2 H, m). 33 ¹H NMR (400 MHz,DMSO-d₆): 8.89 (1 H, br s), 8.23 (1 H, br s), 8.10 (2 H, m), 7.72 (1 H,br m), 6.4-6.8 (2 H, m), 3.78 (3 H, s), 2.65 (3 H, d), 2.36 (3 H, s). 34¹H NMR (400 MHz, DMSO-d₆): 8.07 (1 H, s), 8.05 (1 H, s), 7.97 (1 H, s),6.47 (1 H, d), 6.10 (1 H, m), 6.01 (1 H, d), 3.69 (3 h, s), 2.77 (3 H,d), 2.36 (3 H, s) 35 ¹H NMR (400 MHz, DMSO-d₆): 8.07 (1 H, s), 8.05 (1H, s), 8.01 (1 H, s), 6.63 (1 H, d), 6.16 (1 H, d), 3.70 (3 H, s), 3.19(6H, s), 2.36 (3H, s) 36 ¹H NMR (400 MHz, DMSO-d₆): 8.16 (1 H, s), 8.10(1 H, s), 0.07 (1 H, s), 8.00 (1 H, s), 7.62 (1 H, d), 6.99 (1 H, d),5.68 (1 H, m), 4.45 (1 H, m), 4.21 (2 H, m), 3.78 (4 H, m), 2.57 (3 H,s) 37 ¹H NMR (400 MHz, DMSO-d₆): 8.42 (1 H, br s), 8.16 (1 H, s), 8.10(1 H, s), 8.07 (1 H, s), 7.56 (1 H, d), 7.02 (1 H, d), 3.78 (3 H, s),3.61 (4 H, m), 3.44 (4 H, m), 2.36 (3 H, s) 38 ¹H NMR (400 MHz,DMSO-d₆): 8.68 (1 h, br, s), 8.13 (1 H, s), 8.08 (1 H, s), 8.06 (1 H,s), 7.73 (1 H, d), 6.99 (1 h, br d), 6.89 (1 H, d), 3.75 (3 H, s), 3.72(1 H, m), 2.35 (3 H, s), 1.07 (6 H, d)

Example 3 Comparative Compounds 3.1. Compound A2-[4-[(3-methylbenzimidazol-5-yl)amino]phenyl]acetonitrile

A mixture of Pd₂(dba)₃ (0.01 mmol) and Xantphos (0.01 mmol) in1,4-dioxane (1 mL) was sonicated and added under nitrogen to a mixtureof 6-bromo-1-methyl-benzimidazole (0.45 mmol),2-(4-aminophenyl)acetonitrile (0.58 mmol) and Cs₂CO₃ (0.62 mmol) in1,4-dioxane (2 mL). The mixture was stirred at 110° C. for 12 h. Themixture was diluted (DCM), washed (H₂O), dried (phase separator andconcentrated. The residue was purified by prep HPLC to yield the desiredproduct (Compound A).

MW: 262.3. MS Ms'd: 263.2.

NMR: 1H NMR (400 MHz, DMSO-d6): δ=8.13 (1 H, s), 7.68 (1 H, dd), 7.60 (1H, dd), 7.54 (1 H, d), 7.32 (1 H, d), 7.24 (1 H, t), 6.91 (1 H, dd),3.77 (3 H, s), 3.39 (3 H, s).

3.2. Compound B

A mixture of Pd₂(dba)₃ (0.01 mmol) and Xantphos (0.01 mmol) in1,4-dioxane (1 mL) was sonicated and added under nitrogen to a mixtureof 6-bromo-1-methyl-benzimidazole (0.45 mmol),2,3-dihydro-1,4-benzodioxin-6-amine (0.58 mmol) and Cs₂CO₃ (0.62 mmol)in 1,4-dioxane (2 mL). The mixture was stirred at 110° C. for 12 h. Themixture was diluted (DCM), washed (H₂O), dried (phase separator andconcentrated. The residue was purified by prep HPLC to yield the desiredproduct (Compound B).

MW: 281.3. MS Ms'd: 282.1.

NMR: 1H NMR (400 MHz, DMSO-d6): δ=7.94 (1 H, s), 7.80 (1 H, br s), 7.45(1 H, d), 7.05 (1 H, d), 6.86 (1 H, dd), 6.73 (1 H, dd), 6.61-6.57 (2 H,m), 4.22-4.16 (4 H, m), 3.71 (3 H, s).

3.3. Compound C3-fluoro-4-[methyl-(3-methylbenzimidazol-5-yl)amino]benzonitrile

3.3.1. Step i: 3-fluoro-4-[(3-methylbenzimidazol-5-yl)amino]benzonitrile

A mixture containing 6-bromo-1-methyl-benzimidazole (2.38 mmol),4-amino-3-fluoro-benzonitrile (3.57 mmol), XPhos (0.95 mmol), Cs₂CO₃(7.14 mmol) and Pd(OAc)₂ (0.71 mmol) in dry toluene (8 mL) was stirredat 110° C. for approximately 16 h. The mixture was diluted (EtOAc),washed (H₂O), dried (Na₂SO₄) and concentrated to yield the desiredproduct 3-fluoro-4-[(3-methylbenzimidazol-5-yl)amino]benzonitrile.

3.3.2. Step ii:3-fluoro-4-[methyl-(3-methylbenzimidazol-5-yl)amino]benzonitrile(Compound C)

NaH (7.14 mmol) was added to a solution of3-fluoro-4-[(3-methylbenzimidazol-5-yl)amino]benzonitrile (2.38 mmol) inTHE (10 mL) at 0° C. The mixture was stirred for 30 min. MeI (4.76 mmol)was added and the mixture was stirred at room temperature during 3 h.The mixture was diluted (DCM), washed (H₂O) and concentrated. Theresidue was purified by prep HPLC to yield the desired product (CompoundC).

MW: 280.1. MS Ms'd: 281.0.

NMR: 1H NMR (400 MHz, DMSO-d6): δ=8.13 (1 H, s), 7.68 (1 H, dd), 7.60 (1H, dd), 7.54 (1 H, d), 7.32 (1 H, d), 7.24 (1 H, t), 6.91 (1 H, dd),3.77 (3 H, s), 3.39 (3 H, s).

3.4. Compound D

The synthesis of this compound was described in PCT Int. Appl. (2013) WO2013117645. (Menet et al., 2013)

BIOLOGICAL EXAMPLES Example 4 In Vitro Assays 4.1. JAK1 Inhibition Assay4.1.1. JAK1 Assay polyGT Substrate

Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalognumber 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 isincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20,10 mM MgCl₂, 2 μM non-radioactive ATP, 0.25 ρCi ³³P-gamma-ATP (GEHealthcare, catalog number AH9968) final concentrations) with or without5 μL containing test compound or vehicle (DMSO, 1% final concentration),in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 45 min at 30° C., reactions are stopped by adding of 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionis transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates are washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates is sealed. 40μL/well of Microscint-20 is added, the top of the plates is sealed andreadout is performed using the Topcount (Perkin Elmer). Kinase activityis calculated by subtracting counts per min (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity is determined as:

${{Percentage}\mspace{14mu}{inhibition}} = {\frac{\left( {{{RFU}\mspace{14mu}{test}\mspace{14mu}{compound}} - {{RFU}\mspace{14mu}{control}}} \right)}{\left( {{{RFU}\mspace{14mu}{vehicle}} - {{RFU}\mspace{14mu}{control}}} \right)}*100}$

-   -   RFU test compound=RFU determined for sample with test compound        present    -   RFU control=RFU determined for sample with positive control        inhibitor    -   RFU vehicle=RFU determined in the presence of vehicle

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK1 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM).

4.1.2. JAK1 Ulight-JAK1 Peptide Assay

Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalognumber PV4774) was purchased from Invitrogen. 1 ng of JAK1 was incubatedwith 20 nM Ulight-JAK1 (tyr1023) peptide (Perkin Elmer catalog numberTRF0121) in kinase reaction buffer (25 mM MOPS pH6.8, 0.01% Brij-35, 5mM MgCl₂, 2 mM DTT, 7 μM ATP) with or without 4 μL containing testcompound or vehicle (DMSO, 1% final concentration), in a total volume of20 μL, in a white 384 Opti plate (Perkin Elmer, catalog number 6007290).After 60 min at room temperature, reactions were stopped by adding 20μL/well of detection mixture (1× detection buffer (Perkin Elmer, catalognumber CR97-100C), 0.5 nM Europium-anti-phosphotyrosine (PT66) (PerkinElmer, catalog number AD0068), 10 mM EDTA). Readout is performed usingthe Envision with excitation at 320 nm and measuring emission at 615 nm(Perkin Elmer). Kinase activity was calculated by subtracting relativefluorescence units (RFU) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from RFU obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK1 assay and the calculationof the IC50 for the compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/5 serial dilution, 10 points in afinal concentration of 1% DMSO. When potency of compound seriesincreases, more dilutions are prepared and/or the top concentration arelowered (e.g. 5 μM, 1 μM). The data are expressed as the average IC₅₀from the assays ±standard error of the mean.

TABLE V JAK1 IC₅₀ Values of Illustrative Compounds of the invention Cpd#JAK1 IC₅₀ 1 **** 2 **** 3 **** 4 **** 5 **** 6 **** 7 **** 8 **** 9 ****10 **** 11 **** 12 **** 13 *** 14 **** 15 **** 16 **** 17 ** 18 **** 19**** 20 **** 21 **** 22 **** 23 **** 24 **** 25 **** 26 **** 27 **** 28*** 29 **** 32 *** 33 **** 34 ** 35 * 36 * 37 * 38 **** * >500 nM** >100-500 nM *** >50-100 nM **** 0.1-50 nM

TABLE VI JAK1 IC₅₀ Values of Comparative Compounds Cpd# JAK1 IC₅₀ A *B * C * D *

4.1.3. JAK1 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 1 ng ofJAK1 (Invitrogen, PV4774) is used in the assay. The substrate was 50 nMUlight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121) The reaction isperformed in 25 mM MOPS pH 6.8, 0.01%, 2 mM DTT, 5 mM MgCl2 Brij-35 withvarying concentrations of ATP and compound. Phosphorylated substrate ismeasured using an Eu-labeled anti-phosphotyrosine antibody PT66 (PerkinElmer, AD0068) as described in 1.1.2. Readout is performed on theenvision (Perkin Elmer) with excitation at 320 nm and emission followedat 615 nm and 665 nm.

4.2. JAK2 Inhibition Assay 4.2.1. JAK2 Assay polyGT Substrate

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalognumber PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 isincubated with 2.5 g polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06%Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP(GE Healthcare, catalog number AH9968) final concentrations) with orwithout 5 μL containing test compound or vehicle (DMSO, 1% finalconcentration), in a total volume of 25 μL, in a polypropylene 96-wellplate (Greiner, V-bottom). After 90 min at 30° C., reactions are stoppedby adding of 25 μL/well of 150 mM phosphoric acid. All of the terminatedkinase reaction is transferred to prewashed (75 mM phosphoric acid) 96well filter plates (Perkin Elmer catalog number 6005177) using a cellharvester (Perkin Elmer). Plates are washed 6 times with 300 μL per wellof a 75 mM phosphoric acid solution and the bottom of the plates issealed. 40 μL/well of Microscint-20 is added, the top of the plates issealed and readout is performed using the Topcount (Perkin Elmer).Kinase activity is calculated by subtracting counts per min (cpm)obtained in the presence of a positive control inhibitor (10 μMstaurosporine) from cpm obtained in the presence of vehicle. The abilityof a test compound to inhibit this activity is determined as:

${{Percentage}\mspace{14mu}{inhibition}} = {\frac{\left( {{{RFU}\mspace{14mu}{test}\mspace{14mu}{compound}} - {{RFU}\mspace{14mu}{control}}} \right)}{\left( {{{RFU}\mspace{14mu}{vehicle}} - {{RFU}\mspace{14mu}{control}}} \right)}*100}$

-   -   RFU test compound=RFU determined for sample with test compound        present    -   RFU control=RFU determined for sample with positive control        inhibitor    -   RFU vehicle=RFU determined in the presence of vehicle

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK2 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration is lowered(e.g. 5 μM, 1 μM).

4.2.2. JAK2 Ulight-JAK1 Peptide Assay

Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalognumber PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 wasincubated with 25 nM Ulight-JAK1 (tyr1023) peptide (Perkin Elmer catalognumber TRF0121) in kinase reaction buffer (25 mM HEPES pH7.0, 0.01%Triton X-100, 7.5 mM MgCl₂, 2 mM DTT, 7.5 μM ATP) with or without 4 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 20 μL, in a white 384 Opti plate (Perkin Elmer, catalognumber 6007290). After 60 min at room temperature, reactions werestopped by adding 20 μL/well of detection mixture (1× detection buffer(Perkin Elmer, catalog number CR97-100C), 0.5 nMEuropium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog numberAD0068), 10 mM EDTA). Readout is performed using the Envision withexcitation at 320 nm and measuring emission at 615 nm (Perkin Elmer).Kinase activity was calculated by subtracting relative fluorescenceunits (RFU) obtained in the presence of a positive control inhibitor (10μM staurosporine) from RFU obtained in the presence of vehicle. Theability of a test compound to inhibit this activity was determined as:Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for compound enabling the testing ofdose-response effects in the JAK2 assay and the calculation of the IC₅₀for the compound. Each compound is routinely tested at concentration of20 μM followed by a 1/5 serial dilution, 10 points in a finalconcentration of 1% DMSO. When potency of compound series increases,more dilutions are prepared and/or the top concentration are lowered(e.g. 5 μM, 1 μM). The data are expressed as the average IC₅₀ from theassays ±standard error of the mean.

The following compounds have been tested for their activity against JAK2and the IC₅₀ values, as determined using the assays described herein,are given below in Table VII.

TABLE VII JAK2 IC₅₀ Values of Illustrative Compounds of the inventionCpd# JAK2 IC₅₀ 1 **** 2 **** 3 **** 4 **** 5 **** 6 **** 7 **** 8 **** 9**** 10 **** 11 **** 12 *** 13 ** 14 *** 15 ** 16 **** 17 * 18 **** 19**** 20 *** 21 **** 22 **** 23 *** 24 **** 25 **** 26 *** 27 ** 28 * 29** 32 *** 33 **** 34 * 35 * 36 * 37 * 38 ** * >500 nM ** >100-500 nM*** >50-100 nM **** 0.1-50 nM

TABLE VIII JAK2 IC₅₀ Values of Comparative Compounds Cpd# JAK2 IC₅₀ A *B * C * D *

4.2.3. JAK2 Kd Determination Assay

JAK2 (Invitrogen, PV4210) is used at a final concentration of 5 nM. Thebinding experiment is performed in 50 mM Hepes pH 7.5, 0.01% Brij-35, 10mM MgCl₂, 1 mM EGTA using 25 nM kinase tracer 236 (Invitrogen, PV5592)and 2 nM Eu-anti-GST (Invitrogen, PV5594) with varying compoundconcentrations. Detection of tracer is performed according to themanufacturer's procedure.

4.3. JAK3 Inhibition Assay 4.3.1. JAK3 Ulight-JAK1 Peptide Assay

Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalognumber PV3855) was purchased from Invitrogen. 0.5 ng JAK3 protein wasincubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 10 mM MgCl₂, 2.5mM DTT, 0.5 mM Na₃VO₄, 5 mM b-glycerolphosphate, 0.01% Triton X-100, 1μM non-radioactive ATP, 0.25 Ci ³³P-gamma-ATP (GE Healthcare, catalognumber AH9968) final concentrations) with or without 5 μL containingtest compound or vehicle (DMSO, 1% final concentration), in a totalvolume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom).After 45 min at 30° C., reactions were stopped by adding 25 μL/well of150 mM phosphoric acid. All of the terminated kinase reaction wastransferred to prewashed (75 mM phosphoric acid) 96 well filter plates(Perkin Elmer catalog number 6005177) using a cell harvester (PerkinElmer). Plates were washed 6 times with 300 μL per well of a 75 mMphosphoric acid solution and the bottom of the plates was sealed. 40μL/well of Microscint-20 was added, the top of the plates was sealed andreadout was performed using the Topcount (Perkin Elmer). Kinase activitywas calculated by subtracting counts per min (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity was determined as:

${{Percentage}\mspace{14mu}{inhibition}} = {\frac{\left( {{{RFU}\mspace{14mu}{test}\mspace{14mu}{compound}} - {{RFU}\mspace{14mu}{control}}} \right)}{\left( {{{RFU}\mspace{14mu}{vehicle}} - {{RFU}\mspace{14mu}{control}}} \right)}*100}$

-   -   RFU test compound=RFU determined for sample with test compound        present    -   RFU control=RFU determined for sample with positive control        inhibitor    -   RFU vehicle=RFU determined in the presence of vehicle

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK3 assay and the calculationof the IC₅₀ for each compound. Each compound was routinely tested atconcentration of 20 μM followed by a 1/5 serial dilution, 10 points in afinal concentration of 1% DMSO. When potency of compound seriesincreased, more dilutions were prepared and/or the top concentration waslowered (e.g. 5 μM, 1 μM).

The following compounds have been tested for their activity against JAK3and the IC₅₀ values, as determined using the assays described herein,are given below in Table IX.

TABLE IX JAK3 IC₅₀ Values of Illustrative Compounds of the inventionCpd# JAK3 IC₅₀ 1 *** 2 **** 3 **** 4 ** 5 *** 6 * 7 **** 8 ** 9 ** 10 **11 ** 12 ** 13 * 14 ** 15 * 16 *** 17 * 18 ** 19 *** 20 * 21 *** 22 **23 * 24 ** 25 ** 26 ** 27 ** 28 * 29 * 32 * 33 ** 34 * 35 * 36 * 37 *38 * * >500 nM ** >100-500 nM *** >50-100 nM **** 0.1-50 nM

TABLE X JAK3 IC₅₀ Values of Comparative Compounds Cpd# JAK3 IC₅₀ C * D *

4.3.2. JAK3 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK3(Carna Biosciences, 09CBS-0625B) is used at a final concentration of 10ng/mL. The substrate is Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000(Sigma, P0275) The reaction is performed in 25 mM Tris pH 7.5, 0.01%Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mMb-glycerolphosphate, 10 mM MgCl₂ with varying concentrations of ATP andcompound and stopped by addition of 150 mM phosphoric acid. Measurementof incorporated phosphate into the substrate polyGT is done by loadingthe samples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated ³³P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer).

4.4. TYK2 Inhibition Assay 4.4.1. TYK2 Ulight-JAK1 Peptide Assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalognumber 08-147) was purchased from Carna biosciences. 5 ng of TYK2 wasincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Hepes pH 7.2, 50 mM NaCl, 0.5 mM EDTA, 1mM DTT, 5 mM MnCl₂, 10 mM MgCl₂, 0.1% Brij-35, 0.1 μM non-radioactiveATP, 0.125 μCi ³³P-gamma-ATP (GE Healthcare, catalog number AH9968)final concentrations) with or without 5 μL containing test compound orvehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C.,reactions were stopped by adding 25 μL/well of 150 mM phosphoric acid.All of the terminated kinase reaction was transferred to prewashed (75mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number6005177) using a cell harvester (Perkin Elmer). Plates were washed 6times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates was sealed. 40 μL/well of Microscint-20 was added,the top of the plates was sealed and readout was performed using theTopcount (Perkin Elmer). Kinase activity was calculated by subtractingcounts per min (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the TYK2 assay and the calculationof the IC50 for each compound. Each compound was routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions were prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM).

The following compounds have been tested for their activity againstTYK2; and the IC₅₀ values, as determined using the assays describedherein, are given below in Table XI.

TABLE XI TYK2 IC50 Values of Illustrative Compounds of the inventionCpd# TYK2 IC₅₀ 1 **** 2 **** 3 **** 4 **** 5 **** 6 **** 7 **** 8 **** 9**** 10 **** 11 **** 12 **** 13 **** 14 **** 15 **** 16 **** 17 ** 18**** 19 **** 20 **** 21 **** 22 **** 23 **** 24 **** 25 **** 26 **** 27**** 28 ** 29 ** 32 *** 33 **** 34 * 35 * 36 * 37 * 38 ** * >500 nM** >100-500 nM *** >50-100 nM **** 0.1-50 nM

TABLE XII TYK2 IC₅₀ Values of Comparative Compounds Cpd# TYK2 IC₅₀ C * D*

4.4.2. TYK2 Kd Determination Assay

TYK2 (Carna Biosciences, 09CBS-0983D) is used at a final concentrationof 5 nM. The binding experiment is performed in 50 mM Hepes pH 7.5,0.01% Brij-35, 10 mM MgCl₂, 1 mM EGTA using 50 nM kinase tracer 236(Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) withvarying compound concentrations. Detection of tracer is performedaccording to the manufacturers' procedure.

Example 5 Cellular Assays 5.1. JAK1, JAK2, and TYK2 Selectivity CellAssays 5.1.1. Selective JAK1 Cell Assay, Activation of STAT1 by IFNα inPBMC

Pheripheral blood mononuclear cells (PBMC) are isolated from buffy coatsunder sterile conditions by density gradient centrifugation usingLymphoPrep™ medium (Axis-Shield) followed by 3 subsequent wash steps inPBS without Ca++Mg++. PBMC are resuspended in plain RPMI 1640 mediumcontaining 10% (v/v) heat inactivated FBS, 1% Pen-Strep (100 U/mLPenicilium and 100 μg/mL Streptomycin) and further cultured in ahumidified incubator at 37° C. 5% CO₂.

PBMC are seeded in 24 well plates at 5.0 10⁰⁶ cells/well in a volume of200 μL RPMI 1640 (Invitrogen) containing 10% (v/v) FBS and 1% Pen-Strep(Invitrogen).

PBMC are treated with test compound for 30 min at 37° C. 5% CO₂. 25 μLof 10× concentrated compound dilution is added to the medium. After 30min of test compound/vehicle pre-treatment, PBMC are stimulated for 30min at 37° C. 5% CO₂ with recombinant human IFNα (PeproTech) at finalconcentration of 100 ng/mL by addition of 25 μL (10× concentrated)cytokine trigger to obtain a final volume of 250 μL per well.

All compounds are tested in single starting from 20 μM followed by a 1/3serial dilution, 8 doses in total (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25μM, 0.082 μM, 0.027 μM and 0.009 μM) in a final concentration of 0.2%DMSO.

After 30 min of cytokine stimulation, 250 μL of cell suspension istransferred to a 96-well V-bottom plate, centrifugated for 5 min at 1000rpm to pellet cells, followed by removal of supernatant. The cell pelletis reconstituted in 100 μL 1× Lysis buffer supplemented with EDTA-freeProtease Inhibitor Cocktail (Roche Applied Sciences, Product Number11836170001) followed by sample freezing and storage at −80° C. 1× Lysisbuffer is provided with the Phospho-STAT1 Elisa Kit and containsphosphatase inhibitors. Endogenous levels of phosphorylated STAT1 arequantified using a 96-well PathScan® Phospho-STAT1 (Tyr701) SandwichELISA Kit (Cell Signaling, Product Number #7234) according tomanufacturer's instructions.

HRP activity (HRP is conjugated to the secondary antibody) is measuredby addition of 100 μL of freshly prepared luminol substrate (BMChemiluminescence ELISA Substrate (POD), Roche, Product Number11582950001), incubation for 5 min at room temperature in the dark andmeasured in a Thermo Scientific Luminoskan Ascent Microplate Luminometer(integration time of 200 msec).

5.1.2. Selective JAK2 Cell Assay, Activation of STAT5 by GM-CSF in PBMC

Pheripheral blood mononuclear cells (PBMC) are isolated from buffy coatsunder sterile conditions by density gradient centrifugation usingLymphoPrep™ medium (Axis-Shield) followed by 3 subsequent wash steps inPBS without Ca++Mg++. PBMC are resuspended in plain RPMI 1640 mediumcontaining 10% (v/v) heat inactivated FBS, 1% Pen-Strep (100 U/mLPenicilium and 100 μg/mL Streptomycin) and further cultured in ahumidified incubator at 37° C. 5% CO₂.

PBMC are seeded in 24 well plates at 5.0E06 cells/well in a volume of200 μL RPMI 1640 (Invitrogen) containing 10% (v/v) FBS and 1% Pen-Strep(Invitrogen).

PBMC are treated with test compound by adding 25 μL of 10× concentratedcompound dilution to the medium and incubated for 30 min at 37° C. 5%CO₂. Subsequently, PBMC are stimulated with recombinant human GM-CSF(PeproTech) at final concentration of 0.5 ng/mL by addition of 25 μL(10× concentrated) cytokine trigger per well to obtain a final volume of250 μL. Cells are triggered for 30 min at 37° C. 5% CO₂.

All compounds are tested in single starting from 20 μM followed by a 1/3serial dilution, 8 doses in total (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25μM, 0.082 μM, 0.027 μM and 0.009 μM) in a final concentration of 0.2%DMSO.

After 30 min of cytokine stimulation 250 μL of cell suspension istransferred to a 96-well V-bottom plate following centrifugation for 5min at 1000 rpm to pellet cells. Cell supernatant is removed and pelletis reconstituted in 100 μL 1× Lysis buffer supplemented with EDTA-freeProtease Inhibitor Cocktail (Roche Applied Sciences, Product Number11836170001) followed by sample freezing and storage at −80° C. 1× Lysisbuffer is provided with the Phospho-STAT5 Elisa Kit and containsphosphatase inhibitors. Endogenous levels of phosphorylated STAT5 arequantified using a 96-well PathScan® Phospho-STAT5 (Tyr694) SandwichELISA Kit (Cell Signaling, Product Number #7113) according tomanufacturer's instructions.

HRP activity (HRP is conjugated to the secondary antibody) is measuredby addition of 100 μL of freshly prepared luminol substrate (BMChemiluminescence ELISA Substrate (POD), Roche, Product Number11582950001), incubation for 5 min at room temperature in the dark andmeasured in a Thermo Scientific Luminoskan Ascent Microplate Luminometer(integration time of 200 msec).

5.1.3. Selective TYK2 Cell Assay, Activation of STAT4 by IL-12 in NK-92Cells

NK-92 cells (human malignant non-Hodgkin's lymphoma, interleukin-2(IL-2) dependent Natural Killer Cell line, ATCC #CRL-2407).

NK-92 cells are maintained in Minimum Essential Medium (MEM) Alphamedium w/o ribonucleosides and desoxyribonucleosides, 2 mM L-glutamine,2.2 g/L sodium bicarbonate (Invitrogen, Product Number 22561-021)containing 0.2 mM myo-inositol, 0.1 mM 2-mercapto-EtOH, 0.1 mM folicacid, 12.5% heat inactivated horse serum (Invitrogen, Product Number26050-088), 12.5% heat inactivated FBS, 1% Pen-Strep (100 U/mLPenicilium and 100 μg/mL Streptomycin) and 10 ng/mL recombinant humanIL-2 (R&D Systems). IL-2 is added freshly to the medium with each mediumrefreshment step. Cells are cultured in a humidified incubator at 37° C.5% CO₂.

A subcultured fraction of NK-92 cells are washed once in plain mediumwithout rhIL-2 and seeded in 24-well plates at 0.5E06 cells/well in avolume of 400 μL of plain Alpha MEM medium w/o rhIL-2 containing 0.2 mMmyo-inositol, 0.1 mM 2-mercaptoethanol, 0.1 mM folic acid, 12.5% heatinactivated horse serum (Invitrogen, Product Number 26050-088), 12.5%heat inactivated FBS, 1% Pen-Strep (Invitrogen).

NK-92 cells are treated with test compounds for 30 min prior to rhIL-12stimulation by adding 50 μL of 10× concentrated compound dilution andincubation at 37° C. 5% CO₂. After 30 min of compound/vehiclepre-treatment, cells are stimulated with recombinant human IL-12 (R&DSystems, Product Number 219-IL) at final concentration of 25 ng/mL byaddition of 50 μL (10× concentrated) cytokine trigger to obtain a finalvolume of 500 μL per well. NK-92 cells are triggered with rhIL-12 for 30min at 37° C. 5% CO₂.

All compounds are tested in single starting from 20 μM followed by a 1/3serial dilution, 8 doses in total (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25μM, 0.082 μM, 0.027 μM and 0.009 μM) in a final concentration of 0.2%DMSO.

The levels of phospho-STAT4 in rhIL-12 stimulated NK-92 cells arequantified using a flow cytometric analysis on a Gallios™ flow cytometer(Beckman Coulter). After 30 min of cytokine stimulation the cells arefixed by adding 500 μL of pre-warmed BD Cytofix Fixation Buffer (BDPhosflow™, Product Number 554655) immediately to the wells (fix cellsimmediately in order to maintain phosphorylation state, rather thanspinning down the cells, it is recommended to fix the cells by adding anequal volume of pre-warmed BD Cytofix Buffer to the cell suspension).Cells are incubated for 10 min at 37° C. The fixed cell fraction isresuspended (1 mL) and transferred to FACS tubes followed by acentrifugation step (300×g, 10 min) and removal of the supernatant. Thecell pellet is mixed (vortex) and the cells are permeabilized by adding1 mL of BD Phosflow Perm Buffer III (BD Phosflow™ Product Number 558050)followed by incubation on ice for 30 min. After the permeabilizationstep, the cells are washed twice with BD Pharmingen™ Stain Buffer (BDPharmingen, Product Number 554656) with intermediate centrifugation at300×g for 10 min and removal of the supernatant. The pellet (0.5E06cells) is resuspended in 100 μL of BD Pharmingen™ Stain Buffer andstained by mixing 20 μL of PE Mouse Anti-STAT4 (pY693) to the cells (BDPhosflow™, PE Mouse Anti-STAT4 (pY693), Product Number 558249), thenincubated for 30 min at room temperature in the dark. The stained cellsare washed once with 2 mL of BD Pharmingen™ Stain Buffer and resuspendedin 500 μL of BD Pharmingen™ Stain Buffer and analyzed on a Gallios™ flowcytometer (Beckman Coulter).

For all analyses, dead cells and debris are excluded by forward scatter(FSC) and side scatter (SSC). Changes in phosphorylation of STAT4proteins following cytokine stimulation are approximated by calculatingthe X-median or X-mean fluorescence intensity (MFI) per cell on 100% ofthe gated fraction for all cytokine stimulated, test compound andunstimulated samples.

5.1.4. Results JAK1, JAK2 and TYK2 Assays

Unstimulated samples (no trigger/vehicle (0.2% DMSO) are used as apositive control (100% inhibition). As a negative control (0%inhibition), the stimulated samples (trigger/vehicle (0.2% DMSO)) areused. The positive and negative controls are used to calculate Z′ and‘percent inhibition (PIN)’ values.

Percentage inhibition is calculated from

${{Percentage}\mspace{14mu}{inhibition}} = {\frac{{{RCLU}\left( {{trigger}\text{/}{veh}} \right)} - {{RCLU}\left( {{test}\mspace{14mu}{compound}} \right)}}{{{RCLU}\left( {{trigger}\text{/}{veh}} \right)} - {{RCLU}\left( {{no}\mspace{14mu}{trigger}\text{/}{veh}} \right)}}*100}$wherein

-   RCLU(trigger/veh): Relative Chemilumescent signal determined in    presence of vehicle and trigger-   RCLU(test compound): Relative Chemiluminescent signal determined in    presence of test compounds)-   RCLU(no trigger/veh): Relative Chemiluminescent signal determined in    presence of vehicle without trigger.

In case the readout signal is expressed as X-mean values (flowcytometric analysis of pSTAT4 levels in cytokine stimulated NK-92cells), the RCLU is replaced by X-mean value.

PIN values are plotted for compounds tested in dose-response and EC₅₀values are derived using GraphPad Prism Software applying non-linearregression (sigmoidal) curve fitting.

5.2. JAK1 Mutations in Lung Cancer and Hepatocellular Carcinoma CellLines Assay 5.2.1. JAK1 Mutation Induced Constitutive Signaling

Cancer cell lines with and without JAK1 mutations (Table I—Lung cancercell lines) are cultured with or without serum for 4-6 h, stimulated ornot with a cytokine cocktail (INFγ, IL2, IL4 and IL6) for 5, 10, 30 and45 min. The phosphorylation of JAK1, STAT1, STAT3 and STAT5 areevaluated by immunoblot (Cell Signaling antibodies).

5.2.2. Targeting JAK1 Mutants Using JAK Inhibitors 5.2.2.1. JAK-STATPathway Phosphorylation

Cancer cell lines with and without JAK1 mutations are cultured in thepresence or absence of different concentrations of JAK inhibitors. Cellsare analyzed at 24 and 48 h for effective JAK-STAT pathway inhibition byimmunoblot.

TABLE XIII Table I: Illustrative lung cancer cell lines Present inProtein primary Gene Cell line Tissue Change domain tissue JAK1 NCIH1915Lung I62V FERM — JAK1 SQ1 Lung N226S FERM — JAK1 HCC4006 Lung S383G FERM— JAK1 NCIH2066 Lung L423V Interdomain — (FERM and SH2) JAK1 NCIH1793Lung H525Y SH2 — JAK1 HCC95 Lung N833S Protein Yes kinase 1 JAK1 VMRCLCDLung E223* — — JAK1 NCIH1563 Lung Q161* — — WT A549 Lung — — — JAK1 JAK1U4C Fibrosarcoma — — — −/− *truncation

5.2.2.2. Cell Viability

2D-assay: Cancer cell lines with and without JAK1 mutations are culturedin the presence or absence of increasing concentrations of JAKinhibitors. After 48-72 h, cell viability is measured using the CellTiter-Glo Luminescent cell viability assay (Promega) or MTT assay.Alternatively, cancer cell lines at different culture time points with afix concentration of JAK inhibitor are analyzed for cell viability usingthe Cell Titer-Glo Luminescent cell viability assay (Promega) or MTTassay.

3D-assay: Cancer cell lines with and without JAK1 mutations are seededin semi-solid agar medium. Formation of multi-cellular colonies ismeasured by determining cell viability using a fluorescent dye atdifferent culture time points. Addition of potential inhibitors aftercell seeding allows for the analyses of anti-tumorigenic effects.

5.2.3. Investigating Human JAK1 Mutations in Murine Ba/F3 Cells

(As illustrated in: Kan et al., 2013; Staerk et al., 2005; Zenatti etal., 2011)

Construction of JAK1 expression vectors: Wild type and mutant human JAK1sequences are cloned into retroviral vectors and clones verified bysequencing.

Retroviral infection of Ba/F3 cells: Ba/F3 cells are infected withretroviral supernatants produced in 293T cells.

Ba/F3 cells expressing human WT or mutated JAK1 are cultured with orwithout IL-3 for 4 h and phosphorylation of the JAK-STAT pathwayevaluated by immunoblot.

The transforming potential of JAK1 mutations is assessed by measuringthe ability of each mutation to induce autonomous growth when expressedin cytokine-dependent Ba/F3 cells. Cell growth is assessed in theabsence of the cytokine IL-3.

Mutant JAK1 transduced Ba/F3 cell lines are assessed for theirsensitivity to the JAK inhibitors by culturing them in the presence orabsence of increasing concentrations of JAK inhibitors. After 48-72 h,cell viability is measured using the Cell Titer-Glo Luminescent cellviability assay (Promega) or MTT assay. Alternatively, cancer cell linesat different culture time points with a fix concentration of JAKinhibitor are analyzed for cell viability using the Cell Titer-GloLuminescent cell viability assay (Promega) or MTT assay.

5.2.4. In Vivo Tumorigenic Potential of JAK1 Mutations 5.2.4.1.Xenograft Model

Mutant JAK1 expressing cells are injected subcutaneously in CD1 nu/numice or Rag1−/− mice and evaluated for tumor progression. Subcutaneoustumor volume growth curves are established. The transplantability ofprimary tumors into secondary recipient animals is determined.

5.2.4.2. PDX Model

Patient-Derived Xenografts (PDXs) are based on the transfer of primarytumors (containing JAK1 mutations) directly from the patient into animmunodeficient mouse. To accomplish this, patient tumors must beobtained fresh from surgery, at which point they are mechanically orchemically digested, with a small portion saved as a primary stock andestablished in a NOD-SCID mouse. PDX models are maintained by passagingcells directly from mouse to mouse once the tumor burden becomes toohigh. Tumors can be engrafted heterotopically (implanting tumors intothe subcutaneous flank of a mouse) or orthotopically (directimplantation to the mouse organ of choice).

The phosphorylation of JAK1, STAT1, STAT3 and STAT5 in primary andsecondary tumors are evaluated by immunoblot.

5.3. PBL Proliferation Assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 andproliferation is measured using a BrdU incorporation assay. The PBL arefirst stimulated for 72 h with PHA to induce IL-2 receptor, then theyare fasted for 24 h to stop cell proliferation followed by IL-2stimulation for another 72 h (including 24 h BrdU labeling). Cells arepreincubated with test compounds 1 h before IL-2 addition. Cells arecultured in RPMI 1640 containing 10% (v/v) FBS.

5.4. Human Whole Blood Assay (hWBA) 5.4.1. Protocol 1 5.4.1.1. IL-6Stimulation Protocol

A flow cytometry analysis is performed to establish JAK1 over JAK2compound selectivity ex vivo using human whole blood. Therefore, bloodis taken from human volunteers who gave informed consent. Blood is thenequilibrated for 30 min at 37° C. under gentle rocking, then aliquotedin Eppendorf tubes. Compound is added at different concentrations andincubated at 37° C. for 30 min under gentle rocking and subsequentlystimulated for 20 min at 37° C. under gentle rocking with interleukin 6(IL-6) for JAK1-dependent pathway stimulation or GM-CSF forJAK2-dependent pathway stimulation. Phospho-STAT1 and phospho-STAT5 arethen evaluated using FACS analysis.

5.4.1.2. Phospho-STAT1 Assays 5.4.1.2.1. Preparation of Reagents

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) is diluted 5-foldwith distilled water and pre-warmed at 37° C. The remaining dilutedLyse/Fix buffer is discarded.

10 μg rhIL-6 (R&D Systems, Cat no 206-IL) is dissolved in 1 mL of PBS0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution isaliquoted and stored at −80° C.

A 3-fold dilution series of the compound is prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples are incubated with a 1% final DMSO concentration.

5.4.1.2.2. Incubation of Blood with Compound and Stimulation with IL-6

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μL. Then, 1.5 μL of the test compound dilution isadded to each blood aliquot and the blood samples are incubated for 30min at 37° C. under gentle rocking. One and a half microliter of 10-folddiluted IL-6 stock solution is added to the blood samples (finalconcentration 10 ng/mL) and samples are incubated at 37° C. for 20 minunder gentle rocking.

5.4.1.2.3. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes.

Tubes are centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletis resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100% MeOHis added. Cells are then incubated at 4° C. for 30 min forpermeabilization.

Permeabilized cells are then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μL of 1×PBX containing 3% BSA.

5.4.1.2.4. Cell Labeling with Anti Phospho-STAT1 and Anti-CD4 Antibodies

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. no 612564 and 559319, respectively) andFITC-conjugated anti-CD4 antibody or control FITC-conjugated isotypeantibody are added and mixed, then incubated for 30 min at 4° C., in thedark.

Cells are then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

5.4.1.2.5. Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT1 positive cells aremeasured after gating on CD4⁺ cells, in the lymphocyte gate. Data areanalyzed using the FACSDiva software and the percentage of inhibition ofIL-6 stimulation calculated from the percentage of positive cells forphospho-STAT1 on CD4+ cells.

5.4.1.3. Phospho-STAT5 Assay

Preparation of Reagents

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) is diluted 5-foldwith distilled water and pre-warmed at 37° C. Remaining diluted Lyse/Fixbuffer is discarded.

10 μg rhGM-CSF (AbCys S.A., Cat no P300-03) is dissolved in 100 μL ofPBS 0.1% BSA to obtain a 100 μg/mL stock solution. The stock solution isstored aliquoted at −80° C.

A 3-fold dilution series of the compound is prepared in DMSO (10 mMstock solution). Control-treated samples receive DMSO without the testcompound. All samples are incubated with a 1% final DMSO concentration.

5.4.1.3.1. Incubation of Blood with Compound and Stimulation with GM-CSF

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μL. Then, 1.5 μL of compound dilution is added to eachaliquot and the blood samples are incubated for 30 min at 37° C. undergentle rocking. A 5,000-fold dilution of the GM-CSF stock solution (1.5μL) is added to the blood samples (final concentration 20 pg/mL) andsamples are incubated at 37° C. for 20 min under gentle rocking.

5.4.1.3.2. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes

Tubes are centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletis resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100% MeOHis added. Cells are then incubated at 4° C. for 30 min forpermeabilization.

5.4.1.3.3. Cell labeling with Anti Phospho-STAT5 and Anti-CD33Antibodies

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC mouse anti CD33 antibody (BD Biosciences #345800) or control APCmouse IgG1 isotype antibody (BD Biosciences #345818) are added, mixedthen incubated for 30 min at 4° C., in the dark.

Cells are then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

5.4.1.3.4. Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT5 positive cells aremeasured after gating on CD33⁺ cells. Data are analyzed using theFACSDiva software and correspond to the percentage of inhibition ofGM-CSF stimulation calculated from the percentage of positive cells forphosphor-STAT5 on CD33⁺ cells.

5.5. Protocol 2 5.5.1. Stimulation Protocol

A flow cytometry analysis is performed to establish JAK1 over JAK2compound selectivity ex vivo using human whole blood. Therefore, bloodis taken from human volunteers who gave informed consent. Blood is thenequilibrated for 30 min at 37° C. under gentle rocking, then aliquotedin Eppendorf tubes. Compound is added at different concentrations andincubated at 37° C. for 30 min under gentle rocking and subsequentlystimulated for 20 min at 37° C. under gentle rocking with interleukin 6(IL-6) for JAK1-dependent pathway stimulation, Interferon alpha (IFNα)for JAK1/TYK2 pathway stimulation, interleukin 2 (IL-2) for JAK1/JAK3pathway stimulation or GM-CSF for JAK2-dependent pathway stimulation.Phospho-STAT1 (for IL-6- and IFNα-stimulated cells) and phospho-STAT5(for IL-2- and GM-CSF-stimulated cells) levels are then evaluated usingFACS analysis.

5.5.2. Phospho-STAT Assays 5.5.2.1. Preparation of Reagents

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) is diluted 5-foldwith distilled water and pre-warmed at 37° C. The remaining dilutedLyse/Fix buffer is discarded.

10 μg rhIL-6 (R&D Systems, Cat no 206-IL) is dissolved in 1 mL ofPBS+0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution isaliquoted and stored at −80° C.

10 μg rhIL-2 (R&D Systems, Cat no 202-IL) is dissolved in 1 mL ofPBS+0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution isaliquoted and stored at −80° C.

5 μg rhGM-CSF (AbCys S.A., Cat no P300-03) is dissolved in 12.5 mL ofPBS+0.1% BSA to obtain a 400 ng/mL stock solution. The stock solution isstored aliquoted at −80° C.

A 3-fold dilution series of the compound is prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples are incubated with a 1% final DMSO concentration.

5.5.2.2. Incubation of Blood with Compound and Stimulation with Triggers

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μL. Then, 1.5 μL of the test compound dilution isadded to each blood aliquot and the blood samples are incubated for 30min at 37° C. under gentle rocking. One and a half microliter of 10-folddiluted IL-6 stock solution, 1.5 μL of uIFNα (PBL Biomedical, Cat no11200-1) stock solution, 1.5 μL of 25-fold diluted IL-2 stock solutionor 1.5 μL of 200-fold dilution of the GM-CSF stock solution is added tothe blood samples and samples are incubated at 37° C. for 20 min undergentle rocking.

5.5.2.3. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes.

Tubes are centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletis resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100% MeOHis added. Cells are then incubated at 4° C. for 30 min forpermeabilization.

Permeabilized cells are then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μL of 1×PBX containing 3% BSA.

5.5.2.4. Cell Labeling

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. no 612564 and 559319, respectively) andAPC-conjugated anti-CD4 antibody or control APC-conjugated isotypeantibody (BD Biosciences, Cat. no 555349 and 555751, respectively) areadded to IL-6- and IFNα-stimulated tubes and mixed, then incubated for20 min at 4° C., in the dark.

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC-conjugated anti-CD4 antibody or control APC-conjugated isotypeantibody (BD Biosciences, Cat. no 555349 and 555751, respectively) areadded to IL-2-stimulated tubes, mixed then incubated for 20 min at 4°C., in the dark.

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC mouse anti CD33 antibody (BD Biosciences #345800) or control APCmouse IgG1 isotype antibody (BD Biosciences Cat. no 345818) are added toGM-CSF-stimulated tubes, mixed then incubated for 20 min at 4° C., inthe dark.

Cells are then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

5.5.2.5. Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT1 positive cells aremeasured after gating on CD4+ cells, in the lymphocyte gate for IL-6-and IFNα-stimulated cells. Phospho-STAT5 positive cells are measuredafter gating on CD4+ cells, in the lymphocyte gate for IL-2-stimulatedcells. Phospho-STAT5 positive cells are measured after gating on CD33+cells. Data are analyzed using the FACSDiva software and the percentageof inhibition of IL-6 or IFNα stimulation calculated is from thepercentage of positive cells for phospho-STAT1 on CD4+ cells. For theIL-2 stimulated cells, data are analyzed using the FACSDiva software andthe percentage of inhibition of IL-2 stimulation is calculated from thepercentage of positive cells for phospho-STAT1 on CD4+ cells. For theGM-CSF stimulated cells, the percentage of inhibition of GM-CSFstimulation is calculated from the percentage of positive cells forphosphor-STAT5 on CD33+ cells.

Example 6 In Vivo Models 6.1. CIA Model 6.1.1. Materials

Completed Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)were purchased from Difco. Bovine collagen type II (CII),lipopolysaccharide (LPS), and Enbrel was obtained from Chondrex (Isled'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25 mginjectable syringe, France) Acros Organics (Palo Alto, Calif.),respectively. All other reagents used were of reagent grade and allsolvents were of analytical grade.

6.1.2. Animals

Dark Agouti rats (male, 7-8 weeks old) were obtained from HarlanLaboratories (Maison-Alfort, France). Rats were kept on a 12 hlight/dark cycle (0700-1900). Temperature was maintained at 22° C., andfood and water were provided ad libitum.

6.1.3. Collagen Induced Arthritis (CIA)

One day before the experiment, CII solution (2 mg/mL) was prepared with0.05 M acetic acid and stored at 4° C. Just before the immunization,equal volumes of adjuvant (IFA) and CII were mixed by a homogenizer in apre-cooled glass bottle in an ice water bath. Extra adjuvant andprolonged homogenization may be required if an emulsion is not formed.0.2 mL of the emulsion was injected intradermally at the base of thetail of each rat on day 1, a second booster intradermal injection (CIIsolution at 2 mg/mL in CFA 0.1 mL saline) was performed on day 9. Thisimmunization method was modified from published methods (Jou et al.,2005; Sims et al., 2004).

6.1.4. Study Design

The therapeutic effects of the compounds were tested in the rat CIAmodel. Rats were randomly divided into equal groups and each groupcontained 10 rats. All rats were immunized on day 1 and boosted on day9. Therapeutic dosing lasted from day 16 to day 30. The negative controlgroup was treated with vehicle (MC 0.5%) and the positive control groupwith Enbrel (10 mg/kg, 3× week. s.c.). A compound of interest wastypically tested at 3 doses, e.g. 3, 10, 30 mg/kg, p.o.

6.1.5. Clinical Assessment of Arthritis

Arthritis is scored according to the method of (Khachigian, 2006; Lin etal., 2007; Nishida et al., 2004). The swelling of each of the four pawsis ranked with the arthritic score as follows: 0-no symptoms; 1-mild,but definite redness and swelling of one type of joint such as the ankleor wrist, or apparent redness and swelling limited to individual digits,regardless of the number of affected digits; 2-moderate redness andswelling of two or more types of joints; 3-severe redness and swellingof the entire paw including digits; 4-maximally inflamed limb withinvolvement of multiple joints (maximum cumulative clinical arthritisscore 16 per animal) (Nishida et al., 2004).

To permit the meta-analysis of multiple studies the clinical scorevalues were normalised as follows:

AUC of clinical score (AUC score): The area under the curve (AUC) fromday 1 to day 14 was calculated for each individual rat. The AUC of eachanimal was divided by the average AUC obtained for the vehicle in thestudy from which the data on that animal was obtained and multiplied by100 (i.e. the AUC was expressed as a percentage of the average vehicleAUC per study).

Clinical score increase from day 1 to day 14 (End point score): Theclinical score difference for each animal was divided by the averageclinical score difference obtained for the vehicle in the study fromwhich the data on that animal was obtained and multiplied by 100 (i.e.the difference was expressed as a percentage of the average clinicalscore difference for the vehicle per study).

6.1.6. Change in Body Weight (%) after Onset of Arthritis

Clinically, body weight loss is associated with arthritis (Rall andRoubenoff, 2004; Shelton et al., 2005; Walsmith et al., 2004). Hence,changes in body weight after onset of arthritis can be used as anon-specific endpoint to evaluate the effect of therapeutics in the ratmodel. The change in body weight (%) after onset of arthritis wascalculated as follows:

${Mice}\text{:}\mspace{14mu}\frac{{{Body}\mspace{14mu}{Weight}_{({{week}\; 6})}} - {{Body}\mspace{14mu}{Weight}_{({{week}\; 5})}}}{{Body}\mspace{14mu}{Weight}_{({{week}\; 5})}} \times 100\%$${Rats}\text{:}\mspace{14mu}\frac{{{Body}\mspace{14mu}{Weight}_{({{week}\; 4})}} - {{Body}\mspace{14mu}{Weight}_{({{week}\; 3})}}}{{Body}\mspace{14mu}{Weight}_{({{week}\; 3})}} \times 100\%$

6.1.7. Radiology

X-ray photos were taken of the hind paws of each individual animal. Arandom blind identity number was assigned to each of the photos, and theseverity of bone erosion was ranked by two independent scorers with theradiological Larsen's score system as follows: 0—normal with intact bonyoutlines and normal joint space; 1—slight abnormality with any one ortwo of the exterior metatarsal bones showing slight bone erosion;2—definite early abnormality with any 3 to 5 of the exterior metatarsalbones showing bone erosion; 3—medium destructive abnormality with allthe exterior metatarsal bones as well as any one or two of the interiormetatarsal bones showing definite bone erosions; 4-severe destructiveabnormality with all the metatarsal bones showing definite bone erosionand at least one of the inner metatarsal joints completely erodedleaving some bony joint outlines partly preserved; 5-mutilatingabnormality without bony outlines. This scoring system is a modificationfrom (Bush et al., 2002; Jou et al., 2005; Salvemini et al., 2001; Simset al., 2004).

6.1.8. Histology

After radiological analysis, the hind paws of mice were fixed in 10%phosphate-buffered formalin (pH 7.4), decalcified with rapid bonedecalcifiant for fine histology (Laboratories Eurobio) and embedded inparaffin. To ensure extensive evaluation of the arthritic joints, atleast four serial sections (5 μm thick) were cut and each series ofsections were 100 μm in between. The sections were stained withhematoxylin and eosin (H&E). Histologic examinations for synovialinflammation and bone and cartilage damage were performed double blind.In each paw, four parameters were assessed using a four-point scale. Theparameters were cell infiltration, pannus severity, cartilage erosionand bone erosion. Scoring was performed according as follows: 1-normal,2-mild, 3-moderate, 4-marked. These four scores are summed together andrepresented as an additional score, namely the ‘RA total score’.

6.1.9. Micro-Computed Tomography (μCT) Analysis of Calcaneus (Heel Bone)

Bone degradation observed in RA occurs especially at the cortical boneand can be revealed by μCT analysis (Oste et al., 2007; Sims et al.,2004). After scanning and 3D volume reconstruction of the calcaneusbone, bone degradation is measured as the number of discrete objectspresent per slide, isolated in silico perpendicular to the longitudinalaxis of the bone. The more the bone is degraded, the more discreteobjects are measured. 1000 slices, evenly distributed along thecalcaneus (spaced by about 10.8 μm), are analyzed.

6.1.10. Steady State PK

At day 7 or 11, blood samples were collected at the retro-orbital sinuswith lithium heparin as anti-coagulant at the following time points:predose, 1, 3 and 6 h. Whole blood samples were centrifuged and theresulting plasma samples were stored at −20° C. pending analysis. Plasmaconcentrations of each test compound were determined by an LC-MS/MSmethod in which the mass spectrometer was operated in positiveelectrospray mode. Pharmacokinetic parameters were calculated usingWinnonlin® (Pharsight®, United States) and it was assumed that thepredose plasma levels were equal to the 24 h plasma levels.

6.2. Oncology Models

In vivo models to validate efficacy of small molecules towardsJAK2-driven myleoproliferative diseases are described (Geron et al.,2008; Wernig et al., 2008).

6.3. Mouse IBD Model

In vitro and in vivo models to validate efficacy of small moleculestowards IBD are described (Wirtz and Neurath, 2007).

6.4. Mouse Asthma Model

In vitro and in vivo models to validate efficacy of small moleculestowards asthma are described (Ip et al., 2006; Kudlacz et al., 2008;Nials and Uddin, 2008; Pernis and Rothman, 2002).

6.5. Muarine Model of Psoriatic-Like Epidermal Hyperplasia Induced byIntradermal Injections of IL22 or IL23 6.5.1. Materials

Mouse recombinant IL22 (582-ML-CF), carrier free is provided by R&Dsystems. Mouse recombinant IL23, carrier free (14-8231, CF) is providedby e-Bioscience.

6.5.2. Animals

Balb/c mice (female, 18-20 g body weight) are obtained from CERJ(France). Mice are kept on a 12 h light/dark cycle (07:00-19:00).Temperature is maintained at 22° C., food and water are provided adlibitum.

6.5.3. Study Design

The design of the study is adapted from Rizzo et al, 2011.

On the first day (D1), the mice are shaved around the two ears.

For 4 consecutive days (D1 to D4), the mice received a daily intradermaldose of mouse recombinant IL22 or IL23 (1 μg/20 μL in PBS/0.1% BSA) inthe right pinna ear and 20 μL of PBS/0.1% BSA in the left pinna earunder anesthesia induced by inhalation of isoflurane.

From D1 to D5, mice are dosed with test-compound (10, 30, or 100 mg/kg,po, qd in MC 0.5%), 1 h prior IL23/IL22 injection or with vehicle.

6.5.4. Assessment of Disease

The thickness of both ears is measured daily with an automatic caliper.Body weight is assessed at initiation and at sacrifice. On fifth day, 2hrs after the last dosing, the mice are sacrificed. The pinnae of theear are cut, excluding cartilage. The pinnae are weighed and then,placed in vial containing 1 mL of RNAlater solution or in formaldehyde.

At D4, blood samples are also collected from the retro-orbital sinus forPK profile just before dosing (T0) and 1 h, 3 h, 6 h post-dosing.

There are 8 mice per group. The results are expressed as mean±scm andstatistical analysis is performed using one-way Anova followed byDunnett's post-hoc test versus IL22 or IL23 vehicle groups.

6.5.5. Histology

After sacrifice, ears are collected and fixed in 3.7% formaldehydebefore embedding in paraffin. Two μm thick sections are done and stainedwith hematoxylin and eosin. Ear epidermis thickness is measured by imageanalysis (Sis'Ncom software) with 6 images per ear captured atmagnification ×20. Data are expressed as mean±sem and statisticalanalysis is performed using one-way Anova followed by Dunnett's post-hoctest versus IL22 or IL23 vehicle groups.

6.5.6. RNA extraction, RT-PCR and real-time PCR

IL-17a, IL-22, IL-1β, LCN2 and S100A9 transcript levels in ear tissueare determined using real-time quantitative PCR.

Example 7 Pharmacokinetic, ADME and Toxicity Assays 7.1. ThermodynamicSolubility

The test compound is added to 0.2M phosphate buffer pH 7.4 or 0.1Mcitrate buffer pH 3.0 at a concentration of 1 mg/mL in a glass vial.

The samples are rotated in a Rotator drive STR 4 (Stuart Scientific,Bibby) at speed 3.0 at room temperature for 24 h.

After 24 h, 800 μL of the sample is transferred to an eppendorf tube andcentrifuged 5 min at 14000 rpm. 200 μL of the supernatant of the sampleis then transferred to a MultiscreenR Solubility Plate (Millipore,MSSLBPC50) and the supernatant is filtered (10-12″ Hg) with the aid of avacuum manifold into a clean Greiner polypropylene V-bottom 96 wellplate (Cat no. 651201). 5 μL of the filtrate is diluted into 95 μL (F20)of the same buffer used to incubate in the plate containing the standardcurve (Greiner, Cat no. 651201).

The standard curve for the compound is prepared freshly in DMSO startingfrom a 10 mM DMSO stock solution diluted factor 2 in DMSO (5000 μM) andthen further diluted in DMSO up to 19.5 μM. 3 μL of the dilution seriesas from 5000 μM is then transferred to a 97 μL acetonitrile-buffermixture (50/50). The final concentration range is 2.5 to 150 μM.

The plate is sealed with sealing mats (MA96RD-04S, www.kinesis.co.uk)and samples are measured at room temperature on LC-MS (ZQ 1525 fromWaters) under optimized conditions using Quanoptimize to determine theappropriate mass of the molecule.

The samples are analyzed on LC-MS with a flow rate of 1 mL/min. SolventA is 15 mM ammonia and solvent B is acetonitrile. The sample is rununder positive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column,from Waters. The solvent gradient has a total run time of 2 min andranges from 5% B to 95% B.

Peak areas are analyzed with the aid of Masslynx software package andpeak areas of the samples are plotted against the standard curve toobtain the solubility of the compound.

Solubility values are reported in μM or μg/mL.

7.2. Aqueous Solubility

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.1M phosphate buffer pH7.4or 0.1M citrate buffer pH3.0 at room temperature is added.

The final concentration ranges from 300 μM to 18.75 μM in 5 equaldilution steps. The final DMSO concentration does not exceed 3%. 200 μMPyrene is added to the corner points of each 96 well plate and serves asa reference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 h at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwith a software tool which can be plotted onto a graph. The firstconcentration at which the compound appears completely dissolved is theconcentration reported; however the true concentration lies somewherebetween this concentration and one dilution step higher.

Solubility values measured according to this protocol are reported inμg/mL.

7.3. Plasma Protein Binding (Equilibrium Dialysis)

A 10 mM stock solution of the compound in DMSO is diluted with a factor5 in DMSO. This solution is further diluted in freshly thawed human,rat, mouse or dog plasma (BioReclamation INC) with a final concentrationof 5 μM and final DMSO concentration of 0.5% (5.5 μL in 1094.5 μL plasmain a PP-Masterblock 96 well (Greiner, Cat no. 780285))

A Pierce Red Device plate with inserts (ThermoScientific, Cat no. 89809)is prepared and filled with 750 μL PBS in the buffer chamber and 500 μLof the spiked plasma in the plasma chamber. The plate is incubated for 4h at 37° C. while shaking at 230 rpm. After incubation, 120 μL of bothchambers is transferred to 360 μL acetonitrile in a 96-well roundbottom, PP deep-well plates (Nunc, Cat no. 278743) and sealed with analuminum foil lid. The samples are mixed and placed on ice for 30 min.This plate is then centrifuged 30 min at 1200 rcf at 4° C. and thesupernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201)for analysis on LC-MS.

The plate is sealed with sealing mats (MA96RD-04S) of www.kinesis.co.ukand samples are measured at room temperature on LC-MS (ZQ 1525 fromWaters) under optimized conditions using Quanoptimize to determine theappropriate mass of the molecule.

The samples are analyzed on LC-MS with a flow rate of 1 mL/min. SolventA is 15 mM ammonia and solvent B is acetonitrile. The sample is rununder positive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column,from Waters. The solvent gradient has a total run time of 2 min andranges from 5% B to 95% B.

Peak area from the compound in the buffer chamber and the plasma chamberare considered to be 100% compound. The percentage bound to plasma isderived from these results and is reported as percentage bound toplasma.

The solubility of the compound in the final test concentration in PBS isinspected by microscope to indicate whether precipitation is observed ornot.

7.4. Aldehyde Oxidase Stability

A 10 mM stock solution of test compound in DMSO is first diluted withwater (5 fold) to obtain a 50 μM working solution. A selective inhibitorof aldehyde oxidase (hydralazine) is prepared in water as 5 mM solution.

Incubation mixtures are prepared by adding 10 μL of liver S9 suspension(human and rat, BD Bioscience Gentest, 20 mg/mL) to 86 μL of 50 mMpotassium phosphate buffer, pH 7.4 at 37° C. 2 μL of 5 mM hydralazine isadded (for incubation with the addition of selective inhibitor) or 2 μLof water (for incubation without the addition of the inhibitor).

After 5 min pre-warming, the reaction is initiated by the addition of 2μL of 50 μM test compound to the incubation mixtures. After 0, 3, 6, 12,18, and 30 min of incubation, the reaction (100 μL) is terminated with300 μL of MeCN:MeOH (2:1) with 1% acetic acid mixture containing 10ng/mL of warfarin as analytical internal standard.

Samples are mixed, centrifuged, and the supernatant analysed by LC-MS.

Test compounds are considered as a substrate of aldehyde oxidase ifclearance by S9 is inhibited by hydralazine. Species specific clearanceof test compound may also indicate metabolism by aldehyde oxidase.

Phtalazine is included as a positive control.

The instrument responses (peak area ratio of test compound and internalstandard) are referenced to the zero time-point samples (considered as100%) in order to determine the percentage of compound remaining. Plotsof the percentage of test compounds remaining are used to determine thehalf-life (T_(1/2)) and intrinsic clearance in the S9 incubations usingGraph Pad Prism software.

To calculate the in vitro intrinsic clearance (CL_(int) (μL/min/mg), thefollowing formula is used:

${CLint} = {\frac{0.693}{T\;{1/2}}*\frac{{incubation}\mspace{14mu}{volume}}{{protein}\mspace{14mu}{amount}}*1000}$

7.5. Liver Microsomal Stability

A 10 mM stock solution of compound in DMSO is diluted to 6 μM in a 105mM phosphate buffer, pH 7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-warmed at 37° C.

A Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) workingstock solution of 700 U/mL is diluted with a factor 1:700 in a 105 mMphosphate buffer, pH7.4. A co-factor mix containing 0.528M MgCl₂.6H₂O(Sigma, M2670), 0.528M glucose-6-phosphate (Sigma, G-7879) and 0.208MNADP+ (Sigma, N-0505) is diluted with a factor 1:8 in a 105 mM phosphatebuffer, pH7.4.

A working solution is made containing 1 mg/mL liver microsomes(Xenotech) of the species of interest (human, mouse, rat, dog . . . ),0.8 U/mL G6PDH and co-factor mix (6.6 mM MgCl₂, 6.6 mMglucose-6-phosphate, 2.6 mM NADP+). This mix is pre-incubated for 15min, but never more than 20 min, at room temperature.

After pre-incubation, compound dilution and the mix containing themicrosomes, are added together in equal amount and incubated for 30 minat 300 rpm. For the time point of 0 min, two volumes of MeOH are addedto the compound dilution before the microsome mix is added. The finalconcentration during incubation are: 3 μM test compound or controlcompound, 0.5 mg/mL microsomes, 0.4 U/mL G6PDH, 3.3 mM MgCl₂, 3.3 mMglucose-6-phosphate and 1.3 mM NaDP+.

After 30 min of incubation, the reaction is stopped with 2 volumes ofMeOH.

Of both time points, samples are mixed, centrifuged and the supernatantis harvested for analysis on LC-MS/MS. The instrument responses (i.e.peak heights) are referenced to the zero time-point samples (as 100%) inorder to determine the percentage of compound remaining. Standardcompounds Propanolol and Verapamil are included in the assay design.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 30 min.

7.6. Hepatocyte Stability

Models to evaluate metabolic clearance in hepatocyte are described byMcGinnity et al. Drug Metabolism and Disposition 2008, 32, 11, 1247.

7.7. Caco2 Permeability

Bi-directional Caco-2 assays are performed as described below. Caco-2cells are obtained from European Collection of Cell Cultures (ECACC, cat86010202) and used after a 21 day cell culture in 24-well Transwellplates (Fisher TKT-545-020B).

2×10⁵ cells/well are seeded in plating medium consisting ofDMEM+GlutaMAXI+1% NEAA+10% FBS (FetalClone II)+1% Pen/Strep. The mediumis changed every 2-3 days.

Test and reference compounds (propranolol and rhodamine123 orvinblastine, all purchased from Sigma) are prepared in Hanks' BalancedSalt Solution containing 25 mM HEPES (pH7.4) and added to either theapical (125 μL) or basolateral (600 μL) chambers of the Transwell plateassembly at a concentration of 10 μM with a final DMSO concentration of0.25%.

50 μM Lucifer Yellow (Sigma) is added to the donor buffer in all wellsto assess integrity of the cell layers by monitoring Lucifer Yellowpermeation. As Lucifer Yellow (LY) cannot freely permeate lipophilicbarriers, a high degree of LY transport indicates poor integrity of thecell layer.

After a 1 h incubation at 37° C. while shaking at an orbital shaker at150 rpm, 70 μL aliquots are taken from both apical (A) and basal (B)chambers and added to 100 μL1 50:50 acetonitrile:water solutioncontaining analytical internal standard (0.5 μM carbamazepine) in a 96well plate.

Lucifer yellow is measured with a Spectramax Gemini XS (Ex 426 nm and Em538 nm) in a clean 96 well plate containing 150 μL of liquid frombasolateral and apical side.

Concentrations of compound in the samples are measured by highperformance liquid-chromatography/mass spectroscopy (LC-MS/MS).

Apparent permeability (P_(app)) values are calculated from therelationship:P _(app)=[compound]_(acceptor final) ×V_(acceptor)/([compound]_(donor initial) ×V _(donor))/T _(inc) ×V_(donor)/surface area×60×10⁻⁶ cm/s

V=chamber volume

T_(inc)=incubation time.

Surface area=0.33 cm²

The Efflux ratios, as an indication of active efflux from the apicalcell surface, are calculated using the ratio of P_(app) B>A/P_(app) A>B.

The following assay acceptance criteria are used:

Propranolol: P_(app) (A>B) value ≥20 (×10⁻⁶ cm/s)

Rhodaminc 123 or Vinblastinc: P_(app) (A>B) value <5 (×10⁻⁶ cm/s) withEfflux ratio ≥5.

Lucifer yellow permeability: ≤100 nm/s

7.8. MDCKII-MDR1 Permeability

MDCKII-MDR1 cells are Madin-Darby canine kidney epithelial cells,over-expressing human multi-drug resistance (MDR1) gene, coding forP-glycoprotein (P-gp). Cells are obtained from Netherlands CancerInstitute and used after a 3-4 day cell culture in 24-well Millicellcell culture insert plates (Millipore, PSRP010R5). Bi-directionalMDCKII-MDR1 permeability assay is performed as described below.

3×10⁵ cells/mL (1.2×10⁵ cells/well) are seeded in plating mediumconsisting of DMEM+1% Glutamax-100+1% Antibiotic/Antimycotic+10% FBS(Biowest, S1810). Cells are left in CO₂ incubator for 3-4 days. Themedium is changed 24 h after seeding and on the day of experiment.

Test and reference compounds (amprenavir and propranolol) are preparedin Dulbecco's phosphate buffer saline (D-PBS, pH7.4) and added to eitherthe apical (400 μL) or basolateral (800 μL) chambers of the Millicellcell culture insert plates assembly at a final concentration of 10 μM(0.5 μM in case of amprenavir) with a final DMSO concentration of 1%.

100 μM Lucifer Yellow (Sigma) is added to the all donor buffersolutions, in order to assess integrity of the cell monolayers bymonitoring Lucifer Yellow permeation. Lucifer yellow is a fluorescentmarker for the paracellular pathway and it is used as an internalcontrol in every monolayer to verify tight junction integrity during theassay.

After a 1 h incubation at 37° C. while shaking at an orbital shaker at150 rpm, 75 μL aliquots are taken from both apical (A) and basal (B)chambers and added to 225 μL acetonitrile:water solution (2:1)containing analytical internal standard (10 ng/mL warfarin) in a 96 wellplate. Aliquoting is also performed at the beginning of the experimentfrom donor solutions to obtain initial (Co) concentration.

Concentration of compound in the samples is measured by high performanceliquid-chromatography/mass spectroscopy (LC-MS/MS).

Lucifer yellow is measured with a Fluoroscan Ascent FL Thermo Scientific(Ex 485 nm and Em 530 nm) in a 96 well plate containing 150 μL of liquidfrom all receiver wells (basolateral or apical side).

7.9. Pharmacokinetic Study in Rodents 7.9.1. Animals

Sprague-Dawley rats (male, 5-6 weeks old) are obtained from Janvier(France). Rats are acclimatized for at least 7 days before treatment andare kept on a 12 h light/dark cycle (0700-1900). Temperature ismaintained at approximately 22° C., and food and water are provided adlibitum. Two days before administration of the test compounds, ratsunderwent surgery to place a catheter in the jugular vein underisoflurane anesthesia. After the surgery, rats are housed individually.Rats are deprived of food for at least 16 h before oral dosing and 6 hafter. Water is provided ad libitum.

7.9.2. Pharmacokinetic Study

Compounds are formulated in PEG200/physiological saline (60/40) for theintravenous route and in 0.5% methylcellulose and 10%hydroxylpropyl-β-cyclodextrine pH 3 for the oral route. Test compoundsare orally dosed as a single esophageal gavage at 5 mg/kg under a dosingvolume of 5 mL/kg and intravenously dosed as a bolus via the caudal veinat 1 mg/kg under a dosing volume of 5 mL/kg. Each group consisted of 3rats. Blood samples are collected via the jugular vein with lithiumheparin as anti-coagulant at the following time points: 0.05, 0.25, 0.5,1, 3, 5 and 8 h (intravenous route), and 0.25, 0.5, 1, 3, 5, 8 and 24 h(oral route). Alternatively, blood samples are collected at theretro-orbital sinus with lithium heparin as anti-coagulant at thefollowing time points 0.25, 1, 3 and 6 h (oral route). Whole bloodsamples are centrifuged at 5000 rpm for 10 min and the resulting plasmasamples are stored at −20° C. pending analysis.

7.9.3. Quantification of Compound Levels in Plasma

Plasma concentrations of each test compound are determined by anLC-MS/MS method in which the mass spectrometer is operated in positiveelectrospray mode.

7.9.4. Determination of Pharmacokinetic Parameters

Pharmacokinetic parameters are calculated using Winnonlin® (Pharsight®,United States).

7.10.7-Day Rat Toxicity Study

A 7-day oral toxicity study with test compounds is performed inSprague-Dawley male rats to assess their toxic potential andtoxicokinetics, at daily doses of 100, 300 and 500 mg/kg/day, by gavage,at the constant dosage-volume of 5 mL/kg/day.

The test compounds are formulated in 30% (v/v) HPβCD in purified water.Each group included 5 principal male rats as well as 3 satellite animalsfor toxicokinetics. A fourth group is given 30% (v/v) HPβCD in wateronly, at the same frequency, dosage volume and by the same route ofadministration, and acted as the vehicle control group.

The goal of the study is to determine the lowest dose that resulted inno adverse events being identified (no observable adverse effectlevel—NOAEL).

7.11. Liability for QT Prolongation

Potential for QT prolongation is assessed in the hERG patch clamp assay.

Whole-cell patch-clamp recordings are performed using an EPC10 amplifiercontrolled by Pulse v8.77 software (HEKA). Series resistance istypically less than 10 MΩ and compensated by greater than 60%,recordings are not leak subtracted. Electrodes are manufactured fromGC150TF pipette glass (Harvard).

The external bathing solution contained: 135 mM NaCl, 5 mM KCl, 1.8 mMCaCl₂, 5 mM Glucose, 10 mM HEPES, pH 7.4.

The internal patch pipette solution contained: 100 mM Kgluconate, 20 mMKCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Na₂ATP, 2 mM Glutathione, 11 mM EGTA,10 mM HEPES, pH 7.2.

Drugs are perfused using a Biologic MEV-9/EVH-9 rapid perfusion system.

All recordings are performed on HEK293 cells stably expressing hERGchannels. Cells are cultured on 12 mm round coverslips (German glass,Bellco) anchored in the recording chamber using two platinum rods(Goodfellow). hERG currents are evoked using an activating pulse to +40mV for 1000 ms followed by a tail current pulse to −50 mV for 2000 ms,holding potential is −80 mV. Pulses are applied every 20 s and allexperiments are performed at room temperature.

FINAL REMARKS

It will be appreciated by those skilled in the art that the foregoingdescriptions are exemplary and explanatory in nature, and intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, an artisan will recognize apparent modifications andvariations that may be made without departing from the spirit of theinvention. All such modifications coming within the scope of theappended claims are intended to be included therein. Thus, the inventionis intended to be defined not by the above description, but by thefollowing claims and their equivalents.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication are specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

At least some of the chemical names of compound of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

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The invention claimed is:
 1. A compound according to Formula I:

wherein R¹ is H, or Me; L₁ is —NR²—; —O—, or —CH₂—; Cy is phenyl; R² isH, or C₁₋₄ alkyl; R³ is H, halo, C₁₋₄ alkyl optionally substituted withone or more halo, or C₁₋₄ alkoxy optionally substituted with one or morehalo; R⁴ is H, or halo; R⁵ is —CN, halo, or is -L₂-R⁶; -L₂ is absent, oris —C(═O)—, —C(═O)NR⁷—, —NR⁷C(═O)—, —SO₂NR⁷—, or —NR⁷SO₂—; R⁶ is H, orC₁₋₆ alkyl optionally substituted with one or more independentlyselected R⁸ groups; R⁷ is H, or C₁₋₄ alkyl; R⁸ is OH, CN, halo, or C₁₋₄alkoxy, L_(a) is absent, or is —C(═O)—, —C(═O)O—, or —C(═O)NH—; R^(a)is: H, C₁₋₄ alkyl optionally substituted with one or more independentlyselected R^(b), or C₃₋₇ monocyclic cycloalkyl optionally substitutedwith one or more independently selected R^(c), R^(b) is halo, CN, OH,C₁₋₄ alkoxy, C₃₋₇ cycloalkyl, —SO₂—C₁₋₄ alkyl, or —C(═O)NR^(b1)R^(b2)R^(c) is halo, CN, OH, C₁₋₄ alkyl, —C(═O)OH, or —C(═O)NR^(c1)R^(c2); andeach R^(b1), R^(b2), R^(c1) and R^(c2) is independently selected from H,and C₁₋₄ alkyl, or a pharmaceutically acceptable salt thereof.
 2. Acompound or pharmaceutically acceptable salt according to claim 1,wherein R¹ is Me.
 3. A compound or pharmaceutically acceptable saltaccording to claim 1, wherein the compound is according to Formula IIa:

wherein L₁, R³, R⁴, L_(a), R^(a) and R⁵ are as described in claim
 1. 4.A compound or pharmaceutically acceptable salt according to claim 1,wherein the compound is according to Formula IVa or IVd:

wherein R³, R⁴, R⁵, L_(a), and R^(a) are as described in claim
 1. 5. Acompound or pharmaceutically acceptable salt according to claim 1,wherein the compound is according to Formula Va or Vd:

wherein R³, R⁴, R⁵, L_(a), and R^(a) are as described in claim
 1. 6. Acompound or pharmaceutically acceptable salt according to claim 1,wherein R⁴ is H, F, or Cl.
 7. A compound or pharmaceutically acceptablesalt according to claim 1, wherein R³ is H, Me, or Et.
 8. A compound orpharmaceutically acceptable salt according to claim 1, wherein R⁵ is CN,F, Cl, —SO₂Me or —SO₂Et.
 9. A compound or pharmaceutically acceptablesalt according to claim 1, wherein R^(a) is:


10. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a pharmaceutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toclaim
 1. 11. A compound, or pharmaceutically acceptable salt thereof,according to claim 1 wherein the compound is:N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide,Methyl6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate,N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide,(1R,2R)-N-[6-(4-cyano-2-ethyl-phenoxy)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,(1R,2R)-N-[6-(2-chloro-4-cyano-6-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,(1R,2R)-N-[6-(4-cyano-2-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,(1R,2R)-2-fluoro-N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,(1R,2R)-2-fluoro-N-[6-(2-fluoro-N-methyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide,(1R,2R)-N-[6-(N,2-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,(1R,2R)-N-[6-(4-ethylsulfonyl-N,2-dimethyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide,orN-[6-[4-(cyanomethyl)anilino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide.